Nuclear Localization of Cystatin B, the Cathepsin Inhibitor Implicated in Myoclonus Epilepsy (EPM1)

Massimo Riccio,* Rossella Di Giaimo, Simona Pianetti,* Pier Paolo Palmieri, Marialuisa Melli,,1 and Spartaco Santi*
*Institute of Cytomorphology N.P., C.N.R. c/o Institute Codivilla-Putti-I.O.R., Via di Barbiano 1/10, 40136 Bologna, Italy; and Department of Evolutionary and Experimental Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy

Cystatin B is an anti-protease implicated in myoclonus epilepsy, a degenerative disease of the central nervous system. In vitro, cystatin B interacts with and inhibits proteases of the cathepsin family. Confocal microscopy analysis of the subcellular localization of cystatin B and cathepsin B shows that, in vivo, the two proteins are concentrated in different cell compartments. In fact, cystatin B is found mainly in the nucleus of proliferating cells and both in the nucleus and in the cytoplasm of differentiated cells, while cathepsin B, in either case, is essentially cytoplasmic. However, colocalization of cystatin and cathepsin B is observed in the isolated cell matrix and in the nuclear scaffold of differentiated neuroblastoma cells but not of proliferating cells. This suggests that at least a fraction of cystatin B is bound to the protease in differentiated cells. The electron microscopy analysis of the cell matrix confirms the observation made with confocal microscopy. The cellular activity of cathepsin B was analyzed with a fluorogenic cytochemical assay. A fluorescent signal is observed in the cytoplasm of proliferating cells but is undetectable in the cytoplasm of differentiated cells, suggesting that cathepsin B is active mainly during the cell cycle. This result is consistent with the separate compartimentalization of cystatin B and cathepsin B that we have observed in growing cells. 2001 Academic Press Key Words: EPM1; hereditary neurodegenerative disease; cysteine proteases; cystatin B; protein interaction; differentiation; nuclear matrix; CLSM. INTRODUCTION

Cystatin B is a peptide which is widely distributed in most cell types and tissues that inhibits the cysteine proteases of the cathepsin family [15]. Mutations of the cystatin B gene resulting in loss of function are responsible for UnverrichtLundborg myoclonus epilepsy [68], an inherited, progressive, and lethal autosomic disease that starts between 6 and 15 years of age. Recently, Pennacchio et al. [9] have shown that the knockout of the cystatin B gene generates a neurological disorder in mice, characterized by symptoms very similar to those observed in humans, providing a good animal model for the disease. The main cytological alteration described by the authors is the loss of the cerebellar granule cells, which frequently show apoptotic bodies, chromatin condensation, and other changes typical of programmed cell death [9]. This result suggests that cystatin has an essential role in the cerebellum, protecting granule cells against apoptosis. This function seems to be specific, since in other tissues and cell types the absence of cystatin B does not result in a pathological phenotype. Cystatin B belongs to the cystatin superfamily which includes a large number of proteins, originating from

an ancestral peptide [5, 10, 11]. Cystatins are usually acidic and contain four conserved cysteine residues that form two disulfide bonds. The main structural features conserved in evolution are the type A and B disulfide loop structures, the proline-tryptophane (PW) sequence, and the glutamine-valine-valine-alanineglycine (QVVAG) sequence, corresponding to the consensus QxVxG. This latter sequence and the glycine at position 4 [12] represent the catalytic site where cystatin binds to cathepsin and inhibits its proteolytic function [5, 13]. This control is operated during tissue remodeling events [14], in chronic inflammatory diseases [1518], and during embryo implantation and placentation for normal embryo development [19]. Aberrant regulation of cystatin C associated with increased production and secretion of cysteine proteinases is observed in the invasive phenotype of many metastatic cell types [2023]. The main structural features of cystatin are conserved also in Drosophila where, early in embryogenesis, a cystatin-like zygotic protein of unknown function is expressed at stage 56 in segmentally repeated patterns [24]. Cathepsin B and related proteins are found in the lysosomes [2528]. If the only function of cystatin B were the inhibition of the cathepsin proteases, cystatin
To whom correspondence and reprint requests should be addressed. Fax: 139 051 251208. E-mail: melli@alma.unibo.it. 0014-4827/01 $35.00 84
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Copyright 2001 by Academic Press All rights of reproduction in any form reserved.

Experimental Cell Research 262, 8494 (2001) doi:10.1006/excr.2000.5085, available online at http://www.idealibrary.com on

should colocalize with the proteases of the cathepsin family. Cystatin B does not have a signal peptide and is not a secretory protein, but is synthesized on free ribosomes and cannot enter the rough endoplasmic reticulum, the Golgi, and/or the lysosomes without a carrier. The binding between the cystatin and cathepsin molecules has been studied in vitro [12, 13] while the in vivo colocalization and/or binding of the proteins has not been shown and it is not clear what role the protein has in vivo. Our interest in the molecular basis of myoclonus epilepsy led us to analyze the location of cystatin B and cathepsin B in a variety of cell types, in order to relate the function of the proteins to their compartmentalization. The results confirm that cathepsin B, in proliferating and differentiated cells, is mainly cytoplasmic. In contrast, cystatin B is found both in the nucleus and in the cytoplasm, with a slight prevalence in the nuclear compartment, especially in proliferating cells. A colocalization of some of the cystatin B and cathepsin B molecules is observed in the cellular matrix, but the distribution of the majority of cystatin B does not coincide with that of cathepsin B. These results suggest that cystatin B has multiple function(s), required in the nucleus as well as in the cytoplasm, other than the inhibition of cathepsin.
MATERIALS AND METHODS
Cell culture. Human neuroblastoma SHSY-5Y cells were cultured in Dulbeccos modified Eagles medium (DMEM, Sigma) supplemented with 12% fetal bovine serum (FBS, Euroclone) in a 5%

CO2 atmosphere at 37C. Monkey kidney Cos-1 cells and rat skeletal muscle myoblast L6 cells were cultured in DMEM containing 10% FBS. Human osteosarcoma Saos-2 cells were grown on Iscoves medium (Gibco BRL) supplemented with 10% FBS. SHSY-5Y cells were differentiated for 68 days by the addition of 100 ng/ml of 7S murine nerve growth factor (NGF, Upstate Biotechnology). The medium was changed every two days. When needed, the cells were grown on glass coverslips. Cultures of cerebellar granule cells were obtained from 7-day-old SpragueDawley rat pups. Cerebella were dissected, chopped in small pieces, and treated with 0.025% trypsin/EDTA (Sigma) for 15 min at 37C. Following trypsinization, the tissue was transferred to basal Eagle medium (Gibco BRL) containing 10% FBS and 20 mM KCl. The cells were dissociated by passages through a fire-polished pasteur pipette. The tissue debris were separated from the dissociated cells by passages through a 40-mm nylon cell strainer (Becton Dickinson). The cells, grown for 24 h on glass coverslips, were treated with 3.3 mg/ml aphidilcolin (Sigma), in order to inhibit the proliferation of nonneuronal cells. Cell viability was checked by staining with trypan blue. The medium was changed every 4 days for 89 days of culture. Preparation of the cell matrix and cell fractionation. The preparation of the in situ cell matrix was carried out as previously described [29, 30]. Adherent cells were treated for 10 min at room temperature in TSM buffer (10 mM TrisHCl, pH 7.4, 5 mM MgCl2, 150 mM NaCl) containing 1% Nonidet P-40 (NP-40, Sigma), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml leupeptin, and 10 mg/ml aprotinin. After being washed in TSM buffer, the samples were incubated with 30 U/ml of DNAse I (Sigma) in TSM buffer for 40 min at room temperature. The cells were then washed in TSM buffer and extracted twice for 5 min with TBS containing 2 M NaCl. Finally the samples were washed in phosphate-buffered saline (PBS; pH 7.4) and fixed for 20 min at 4C in PBS containing 4% paraformaldehyde. The protein extract used in the Western blot analysis was obtained from the cell fractions and concentrated by trichloroacetic acid precipitation. The total cell extract used in the Western blot analysis was obtained as follows. The cells were washed in PBS at pH 7.4 and incubated 10 min at 4C in 10 mM TrisHCl, pH 7.8, containing 1% NP-40, 10 mM b-mercaptoethanol, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml soybean trypsin inhibitor, 15 mg/ml calpain inhibitor I, and 7 mg/ml calpain inhibitor II (Boehringer Mannheim, Germany). The cells were disrupted by 50 passages through a 25-gauge needle and the homogenate was sonicated for 1 min at 4C and centrifuged for 10 min at 48,000g. The protein concentration was measured by the Bio-Rad protein assay. Immunofluorescence and confocal microscopy. The cells were fixed for 30 min at 4C in PBS containing 4% paraformaldehyde and treated for 10 min with PBS containing 0.2% Triton X-100 followed by 30-min incubation at room temperature in PBS containing 3% bovine serum albumin (BSA). The anti-cathepsin B and anti-cystatin B polyclonal antibodies (Abs; Biogenesis) were diluted 1:50 in PBS containing 3% BSA and incubated with the cells or the matrix for 1 h at room temperature. After being washed in PBS containing 3% BSA, the samples where incubated for 1 h at room temperature with a FITC-conjugated goat anti-rabbit Ab (F(ab9)2 fragment; Sigma) or with a FITC-conjugated donkey anti-sheep Ab (Sigma) to reveal the antigenantibody interaction. For double immunofluorescence the primary Abs were detected by using a Cy5-conjugated donkey antirabbit Ab (Jackson) and a FITC-conjugated donkey anti-sheep Ab (Sigma). The cystatin A and B proteins used for the experiments of Fig. 1A were from Biogenesis; the cathepsin B and D proteins were from Sigma. The confocal imaging was carried out with a Radiance 2000 confocal laser scanning microscope (Bio-Rad), equipped with a Nikon 40X, 1.4 N.A., and with krypton and red diode lasers. To detect the FITC fluorescence and the fluorogenic cathepsin B activity, the samples were excited with the 488-nm line of the krypton laser. The laser beam was attenuated at 10% with a neutral density filter and the emission signal was detected after passage through a long-pass filter (LP: 500 nm) by a photomultiplier tube. Pseudo-phase-contrast images of the sample were simultaneously detected. To detect FITC and Cy5 together, the samples were simultaneously excited with the 488-nm line of the krypton laser and the 637-nm line of the red diode laser, respectively. The emission signals

from FITC and Cy5 were separated by a dichroic mirror (DM: 560 nm) and simultaneously detected by two photomultiplier tubes. Two barrier filters (BP: 515/30 nm for FITC and LP: 660 nm for Cy5) were placed in front of the two photomultiplier tubes to minimize the overlapping between the two signals. Optical sections were obtained at increments of 0.3 mm in the Z-axis and were digitized with a scanning mode format of 512 3 512 pixels and 256 gray levels. The image processing, the volume rendering, and the quantitative analysis were performed using ImageSpace software (Molecular Dynamics, Inc.) running on a Indigo workstation (Silicon Graphics, Mountain View, CA). The evaluation of the colocalized fluorochromes was performed by LaserPix software (Bio-Rad) as previously described [31, 32]. For the quantitative analysis, 50 randomly chosen cells were analyzed and the fluorescence intensity was quantified on single optical sections passing through the nucleus of each cell. The nuclear and the cytoplasmic fluorescence intensities were evaluated on each cell by measuring the mean pixel intensity in a fixed area of 466 pixels (;50 mm2 with a 60X objective lens). The mean of these values was determined for each experimental condition. The statistical signifiNUCLEAR LOCALIZATION OF CYSTATIN B 85 cance of the differences between the experimental points was evaluated by Students t test. Cathepsin B activity assay. The activity assay was carried out according to Kayser et al. [33]. The cells were fixed in 1% paraformaldehyde in PBS (pH 7.4) for 15 min at 4C. After being washed in 0.1 M phosphate buffer (PB, pH 6.2), the samples were incubated for 15 min at 37C in 0.1 M PB, pH 6.2, containing 1 mg/ml N-CBZ-alaargarg-4-methoxy-2-naphthylamide (Bachem), 1 mM 2-hydroxy-5nitrobenzaldehyde (Merck), 1 mM dithiothreitol (Merck), 1.3 mM Na2 EDTA, 2.67 mM L-cysteine (Sigma). After the incubation, the cells were mounted in 1,4-diazabicyclo-[2.2.2]octane (Sigma) and observed in confocal microscopy. The specificity of the reaction was tested by using 5 mM leupeptin (Sigma) and/or 0.05 mg/ml egg white cystatin (Sigma). Electron microscopy. The cell pellets were fixed in 1% glutaraldehyde in 0.1 M PB for 30 min at 4C and embedded in Epon 812 resin (Electron Microscopy Science, PA). Following a preincubation in 0.05 M TrisHCl, pH 7.6, 0.14 M NaCl, 5% goat serum, 0.1% BSA, the cell thin sections were incubated for 1 h at room temperature with the primary Ab diluted 1:10 in the same buffer. After being washed in 0.05 M TrisHCl, pH 7.6, 0.14 M NaCl, and 0.1% BSA, the samples were incubated for 1 h at room temperature with 10-nm gold-conjugated secondary Ab (anti-rabbit Ab for anti-cystatin B primary Ab or rabbit anti-sheep Ab for anti-cathepsin B primary Ab) diluted 1:10 in 0.02MTrisHCl, pH 8.2, 0.14MNaCl, and 0.1% BSA. The amplification with a Silver Enhancer kit (Amersham) was carried out for 3 min at room temperature and counterstained with uranyl acetatelead citrate. When double labeling was utilized, one side of the grid was incubated with anti-cystatin B antibodies (5-nm colloidal gold particles) while the opposite site was incubated with anti-cathepsin B antibodies (15-nm colloidal gold particles). Incubation of the grids with gold-conjugated secondary antibodies only was used as control. To control the background level, preabsorption experiments with the cystatin B and cathepsin B proteins or with the nonspecific proteins cystatin A and cathepsin D were also carried out. The number of gold particles per square micrometer in the cytoplasmic and nuclear compartments was evaluated. For each microphotograph, the background was subtracted from the values calculated in each cellular compartment. The statistical significance was evaluated by Students t test. Western blot analysis. Fifteen micrograms of proteins was separated on 15% SDSPAGE and electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated for 1 h at room temperature in PBS containing 0.1% Tween 20, 0.2% BSA, and 3% dry milk. Primary Abs (cystatin B polyclonal Ab or anti cathepsin B polyclonal Ab) diluted 1:2000 were added and incubated for 1 h at room temperature. After being washed, the strips were incubated with horseradish peroxidase (HRP) conjugated secondary Abs: donkey anti-rabbit Ab (Amersham) for anti-cystatin B primary Ab or donkey anti-sheep Ab (Sigma) for cathepsin B primary Ab. The HRP conjugated secondary Abs were detected using the ECL kit (Amersham).

RESULTS

Intracellular localization of cathepsin B and cystatin

B in tissue culture cell lines. We have analyzed the subcellular localization of cystatin B and cathepsin B in a number of nondifferentiated established and primary cell lines during proliferation. The cells, immunostained with polyclonal antibodies against the cystatin B and the cathepsin B proteins, were analyzed by confocal microscopy. The specificity of the immunoreaction was tested using immunofluorescence confocal microscopy by preabsorption of the polyclonal antibodies with specific and with unrelated proteins (Fig. 1A). The preabsorption of cystatin B and cathepsin B antibodies with the respective proteins inhibits the binding of the antibody to the cellular antigen (Figs. 1Aa and 1Ab), while antigen antibody interaction is still observed after preabsorption of anti-cystatin B antibodies with the unrelated cystatin A and cathepsin D proteins (Figs. 1Ac and 1Ad). We have also tested the possibility of a cross reaction between anti-cystatin B and anti-cathepsin B antibodies with other members of the cystatin and cathepsin families of proteins without observing an
FIG. 1. Specificity of antibodies against cystatin B and cathepsin B. (A) SHSY-5Y cells 1 anti-cystatin B (a), anti-cathepsin B (b) antibodies preadsorbed with the specific antigens or with cystatin A (c) and cathepsin D (d). (B) Western blot analysis of total cell extracts from SHSY-5Y (lanes 1 and 3) and Cos-1 (lanes 2 and 4) cells, after electrophoresis on a 15% SDSPAGE. 86 RICCIO ET AL.

interaction. The incubation of cells with the secondary antibody alone does not show background fluorescence either (data not shown). The specificity of the antibodies is also confirmed by Western blot analysis, shown in Fig. 1B. Lanes 1 and 2 show the interaction of anticystatin B antibodies with protein extracts from SHSY-5Y and Cos-1 cells, respectively, while lanes 3 and 4 show the binding of the same cell extracts with anti-cathepsin B antibodies. Only one band is detectable in each lane, corresponding to the molecular weight of cystatin B (12 kDa, lanes 1 and 2) and cathepsin B (25 kDa, lanes 3 and 4).

Figure 2 shows the localization of cystatin B in a number of different cell types. The immunofluorescence analysis was carried out on proliferating human neuroblastoma SHSY-5Y (Figs. 2A2D) cells, monkey kidney Cos-1 cells (Figs. 2E2H), human osteosarcoma Saos-2 cells (Figs. 2I2L), and rat myoblasts L6 cells from skeletal muscles (Figs. 2M2P). The comparison between the fluorescent and phase contrast images allows a precise definition of the antigenantibody interaction and locates the cystatin B mainly in the nucleus of all cell types analyzed. In contrast, cathepsin B is mainly cytoplasmic, with a diffused distribution in the SHSY-5Y cells and a granular distribution in the Cos-1, Saos-2, and L6 cells. In Cos-1 and L6 cells the fluorescence is concentrated in the perinuclear region where the Golgi cisternae are also located. To establish whether the distinct cellular distribution of the two proteins is unique to growing cells, we have compared the subcellular distribution of cystatin B and cathepsin B in growing (Fig. 3Aa3Ad) and differentiated (Fig. 3Ae3Ah) SHSY-5Y neuroblastoma cells. Differentiation induced by the NGF is indicated by the presence of neurite extensions, shown by the phase contrast images (Figs. 3Af and 3Ah). The comparison of the fluorescence intensity of Figs. 3Aa and 3Ae suggests that, in growing cells, cystatin B is mainly nuclear while in differentiated cells it is abundant both in the nucleus and in the cytoplasm. Cathepsin B maintains essentially the same cytoplasmic distribution in proliferating and differentiated cells (Figs. 3Ac and 3Ag). The histogram of Fig. 3B gives a measure of the signal intensity of the anti-cystatin B antibodies in the nucleus and in the cytoplasm of growing and differentiated (1NGF) cells and confirms that the

FIG. 2. Immunocytochemical localization of cystatin B (A, E, I, M) and cathepsin B (C, G, K, O) in proliferating cells: human neuroblastoma cell lines SHSY-5Y (AD), monkey kidney Cos-1 cells (EH), human osteosarcoma Saos-2 cells (IL), and rat skeletal muscle myoblast L6 cells (MP). Confocal microscopy image analysis: A, C, E, G, I, K, M, O; Phase contrast: B, D, F, H, J, L, N, P. NUCLEAR LOCALIZATION OF CYSTATIN B 87

fluorescence is higher in the nucleus compared with the cytoplasm. In addition, the amount of cystatin B seems higher in differentiated cells and this is confirmed by the Western blot analysis of Fig. 3C.

The intensity of the antibody staining of the bands migrating in the gel at a position corresponding to that of cystatin B is higher in the differentiated compared to the growing cells. Further evidence on the cellular distribution of cystatin B in the SHSY-5Y neuroblastoma cells was obtained by electron microscopy (EM) immunogold labeling (Fig. 4). The gold particles are observed in the nucleus and in the cytoplasm of growing (Fig. 4A) and differentiated (Fig. 4B) cells. The nuclear labeling is distributed on the nucleoplasm and on the interchromatin granules. The cytoplasmic labeling is also present in the mitochondria [25, 26]. Table 1 shows the grain density distribution in the nuclear and cytoplasmic compartments of SHSY-5Y neuroblastoma cells. The difference of nuclear/cytoplasmic distribution of gold particles shown by EM analysis is significant, but smaller than that shown by immunofluorescence analysis. This is possibly due to the intrinsic differences of the two techniques. In fact, the immunofluorescence technique involves permeabilization of the cell, leaving the antigen exposed and available for binding to the antibodies. In contrast, the immunogold labeling technique requires ultrathin sections, where the antigen is available mainly on the surface of the slice and the efficiency of labeling changes depending on the compartimentalization of the antigen itself [27].

In agreement with our observations, the immunogold labeling of cathepsin B is similar in proliferating and differentiated cells in both cellular districts (data not shown). Localization of cystatin B in primary cerebellar granule cells. As myoclonous epilepsy correlates with mutation( s) of the cystatin B gene and with cerebellar damage [9], we have studied the subcellular distribution of cystatin B and cathepsin B in rat primary cerebellar granule cells (Fig. 5) cultured for 2 (Figs. 5A
FIG. 3. Immunocytochemical localization of cystatin B and cathepsin B in differentiated SHSY-5Y cells. (A) Proliferating human neuroblastoma cells (ad); NGF differentiated cells (e h). Phase contrast images (b, d, f, h); fluorescent images (a, c, e, g). The intensity of fluorescence is represented on a pseudocolor scale (palette bar to the right of g). (B) Histograms showing the quantitation by confocal microscopy of the subcellular distribution of cystatin B in proliferating and differentiated SHSY-5Y. The average nuclear (n) and cytoplasmic (h) values are shown. (C) Western blot analysis of proliferating (lane 1) and differentiated (lane 2) SHSY-5Y cell extracts stained with anti-cystatin B antibodies. Equal amounts of total cell protein extracts were loaded on each slot. 88 RICCIO ET AL.

5D), 4 (Figs. 5E5H), and 9 (Figs. 5I5L) days. The phase contrast images (Figs. 5B, 5F, and 5J and 5D, 5H, and 5L) show the neurite extensions typical of the in vitro process of differentiation of the cells which occurs in about 9 days. The signal due to the anticystatin B antibodies is strong and mainly nuclear, already 2 days after the start of the culture, when the cells are not yet differentiated (Figs. 5A and 5B). As the time of culture progresses and the cells undergo differentiation, the intensity of the signal increases both in the nucleus and in the cytoplasm (Figs. 5E and 5F; 5I and 5J). The anti-cathepsin B antibody staining indicates a cytoplasmic distribution of the protease already present in cells cultured for 2 days. This set of data is in agreement with the results obtained with all established cell lines previously analyzed, suggesting that the cellular distribution of the two proteins is similar in most cell types, including the cerebellar granule cells. Localization of cystatin B in the cell matrix. Although most of the cystatin B and cathepsin B does not colocalize, at least a fraction of the two proteins should be functionally associated. The structural relationship between the cell compartments and proteins can be studied by EM and/or confocal microscopy on the in situ isolated cell matrix, which uncovers cytoskeletal elements, nuclear lamina, inner nuclear matrix, and nucleolar remnants. Figure 6A shows the confocal microscopy analysis of the cell matrix from proliferating SHSY-5Y (Figs. 6Aa and 6Ab) and Cos-1 (Figs. 6Ac and 6Ad) cells immunostained with the anti-cystatin B (Figs. 6Aa and 6Ac) and the anti-cathepsin B (Figs. 6Ab and 6Ad) antibodies. Fluorescence is clearly detectable in the nuclear and perinuclear regions of the cells stained with anti-cathepsin antibodies. The cells stained with anti-cystatin antibodies do not show fluorescence in the nucleus or in the cytoplasm of either cell type. The Western blot analysis of the protein extracts from the matrix of the SHSY-5Y cells confirms the results (Fig. 6B).'

The immunoreactive bands corresponding to cathepsin B and cystatin B are both present in the soluble fraction (lane 1). In contrast, only one faint band migrating to the position of cathepsin B is present in lane 2, while the cystatin B band is not detectable. Antibodies against the soluble protein GAPDH were used as control to test the purity of the cell fractionation procedure. GAPDH staining is present only in the soluble fraction of the cells and is absent in the matrix fraction. These results suggest that, in proliferating cells, part of the cathepsin B is associated with the nuclear scaffold and part of it with the cytoskeletal proteins. Similar results were obtained from Cos-1 cells (data not shown). Cystatin B is evidently soluble and is probably lost during the extraction procedure. The same type of analysis was carried out on the cell

FIG. 5. Immunofluorescence confocal microscopy of primary cerebellar granule cells. The intensity of fluorescence is represented in pseudocolor scale as in Fig. 3A. Differentiating granule cells stained with anti-cystatin B and anti-cathepsin B antibodies were simultaneously analyzed by confocal and phase contrast images A/B; C/D; E/F; G/H; I/J; K/L. NUCLEAR LOCALIZATION OF CYSTATIN B 89

matrix of NGF-treated SHSY-5Y cells (Fig. 7).

Both anti-cystatin and anti-cathepsin antibodies label the nucleus strongly and the cytoplasm of the neuroblastoma cells weakly. The signals, indicated by the red

and green colors, colocalize also in the cytoplasmic granules. The merging of the two colors results in a bright yellow, which indicates almost perfect coincidence of the two signals (Fig. 7C) in the cytoplasm and in the nucleus. In fact, the scatter plot (Fig. 7D) obtained from the individual analysis of the optical doubledetection shows that more than 90% of the red overlaps the green signal. The cellular matrix was further studied by EM (Fig. 7E). The inner nuclear matrix appears as a network of granulofilamentous structures connecting the residual nucleolar remnants; the nuclear lamina, in cross sections, reveals the nuclear pore complexes. The residual cytoskeleton is mainly constituted by threads of long filaments surrounding the nuclear matrix, possibly corresponding to intermediate filaments. Figure 7E shows a detail of a section of the SHSY-5Y cells double labeled with anti-cystatin B (5 nm) and anti-cathepsin B (10 nm) polyclonal antibodies by the immunogold method. The signals are both localized near the ribonucleoprotein particle cluster along the filaments of the inner nuclear matrix. Moreover, gold particles are associated with the filamentous components of the cytoskeleton. This result indicates that cystatin B, soluble in growing cells, is partly bound to the cell matrix, while in differentiated cells it is associated to the cathepsin B at specific sites of the insoluble inner nuclear matrix components. Intracellular cathepsin B activity in proliferating and differentiated cells. Although cystatin B and cathepsin B are both found in the nucleus of proliferating cells, they may not interact. However, in the cytoplasm, cystatin B could be bound to cathepsin B and fulfill its role as inhibitor. Thus, we have analyzed in SHSY-5Y and in rat primary cerebellar granule cells the activity of cathepsin B using a fluorogenic cytochemical assay (Bachem Biochemica, Heidelberg). The intracellular activity of cathepsin B was monitored by the hydrolysis of the synthetic substrate Z-Arg-Arg-4M bNA. The released 4M bNA produces a fluorogenic signal which is detected by confocal microscopy (Fig. 8). Two cathepsin protein specific inhibitors were used as controls: cystatin B and leupeptin. In this test the inhibition of cathepsin activity is revealed by the absence of fluorescence. For both specific inhibitors no signals were detected (data not shown). In growing cells, the enzymatic activity of cathepsin B is detectable both in the nucleus and in the cytoplasm, with a distribution similar to that of cathepsin itself (Figs. 8A
TABLE 1 Quantitation of the Cytoplasmic and Nuclear Mean Grain Density of Proliferating and Differentiated Human SHSY-5Y Neuroblastoma Cells Stained with Anti-Cystatin Antibodies
Nucleus Cytoplasm SHSY-5Y 8.1 6 0.6 6.3 6 0.6 SHSY-5Y 1 NGF 9.8 6 1.0 8.1 6 0.9 Note. Values are expressed as number of gold particles/mm2 6 SD. The comparison is between the number of cytoplasmic and nuclear gold particles. n 5 80; Students t test: P , 0.01.

FIG. 4. Electron microscopy immunogold localization with anticystatin B antibodies on a thin section of proliferating (A) and differentiated

(B) SHSY-5Y neuroblastoma cells. N and C indicate the nuclear and the cytoplasmic compartments, respectively. (C) The antibodies were preabsorbed with cystatin B protein before the immunogold labeling. The background of growing and differentiated cells was the same. Bar, 1 mm. 90 RICCIO ET AL.

and 8E).

However, while in growing SHSY-5Y cells the fluorescence is uniformly diffused on the entire nuclear and cytoplasmic region, in CGC cells is concentrated in granular cytoplasmic structures. In differentiated neuroblastoma cells (Fig. 8C), and in rat primary cerebellar granule cells after 9 days of in vitro culture (Fig. 8G), cathepsin B, although present, is inactive. This suggests that, in proliferating cells, cystatin B is not bound to and does not inhibit cathepsin while, in differentiated cells, it becomes associated with and inhibitory to the cystein protease activity. This observation is in agreement with the presence of cystatin B in the nuclear matrix of differentiated neuroblastoma cells.
DISCUSSION

The results described in this manuscript show, for the first time, that cystatin B, an anti-protease of the stefin family of proteins, is mostly localized in the cell nucleus, where it may fulfill a yet unknown function. We also show that at least part of the cellular cystatin B and cathepsin B, in differentiated cells, is bound to the cell matrix and may interact. Furthermore, in the neuroblastoma and cerebellar granule cells, we find a correlation between the increased cytoplasmic localization of cystatin B and the differentiation of the cell. This suggests a role of cystatin B as inhibitor and regulator of proteases during cell differentiation. Calkins et al. [28] have recently studied, by confocal microscopy, the immunocytochemical localization of the cysteine protease inhibitors and cysteine proteases in two cell lines derived from murine hepatoma and embryonic liver. Their results suggest that cystatin A and B are associated with vescicular membranes and membrane fractions of the cells and cystatin A, in

particular, is present on the surface of hepatocytes but not of hepatoma cells. Cathepsins B, H, and L are all cytoplasmic but localized in vescicles and structures associated with the bottom surface of these cells. In conclusion, cystatin and cathepsins are present in different compartments of the cytoplasm. An immunocytochemical localization of the proteins of the cathepsin family in granular cytoplasmic structures of rat kidney cells was also reported by Matsuba et al. [26], suggesting a role of the cysteine proteases in the processing of renin. Thus, although cystatins and cathepsins interact in vitro, it is not clear to what extent they interact in vivo. In fact the existing evidence is for their presence in separate cell compartments. Our results differ in some respects from those reported by Calkins et al. [28], since cystatin A (not shown) and B show a very similar nuclear localization in all cells presented in this work and in the following cell types (not shown): Morris hepatoma cells, primary rat oligodendrocytes and astrocytes, and two different rat and human neuroblastoma cell lines. Cystatins A and B are also present in the cytoplasm, especially in differentiated cells, but most of the protein is not membrane bound, which is consistent with the absence of an aminoterminous hydrophobic signal in the peptide sequence. Of course, we cannot exclude the existence of a very small amount of protein that is membrane bound. Cystatin B seems to be soluble and, although its sequence does not contain an obvious nuclear localization signal, the nuclear concentration of the protein in growing cells is higher than the cytoplasmic concentration. This may be due to a carrier that allows the translocation of cystatin into the nucleus or to the interaction of cystatin with a nuclear protein(s) or structure, which causes the retention of the anti-protease. The nuclear localization is very clear also in the cerebellar granule cells and in their progenitors and this may be of importance to myoclonus epilepsy, where the absence of cystatin B seems to result in the apoptosis of many granule cells [9]. On the other hand, the presence of cystatin B in mitochondria revealed by the EM analysis may also correlate with an antiapoptotic function of the anti-protease. The cytoplasmic localization of cathepsin B is consistent with that found by other authors, and the granular texture of the cytoplasmic staining in Fig. 2 is
FIG. 6. Immunofluorescence confocal microscopy (A) of the SHSY-5Y (a, b) and Cos-1 (c, d) cell matrix. Anti-cystatin B antibodies (a, c); anti-cathepsin B antibodies (b, d). Western blot analysis (B) of the soluble fraction (lane 1) and of the cell matrix (lane 2) from SHSY-5Y cells. The protein extracts were obtained from the same cell preparation. The soluble protein was used as control. NUCLEAR LOCALIZATION OF CYSTATIN B 91

consistent with a localization of the protease in vesicular structures, i.e., Golgi apparatus/lysosomes. We have also examined the localization of cathepsins D, L, and H (not shown), with results similar to those of cathepsin B. The different cellular localization of cystatin B that we have observed compared to other authors may be specific to the cell type and reflect specific functions.

In the cell matrix, cathepsin B is partly bound and, in proliferating neuroblastoma cells, is present both in the cytoplasm and in the nucleus, while cystatin B is not detectable, suggesting that it is solubilized by the matrix extraction procedure. In contrast, in differentiated SHSY-5Y neuroblastoma cells, cathepsin B and cystatin B are both detectable, bound to the nuclear and, to some extent, to the cytoplasmic matrix and the confocal microscopy analysis shows perfect coincidence of their fluorescence signals. This is consistent with the EM analysis, where the 5- and 10-nm gold particles are near each other. The results suggest the cellular interaction of at least a fraction of the cystatin and cathepsin proteins at the level of the cell matrix. In agreement with this finding is the result of the experiment of Fig. 8, which shows that cathepsin B is active in proliferating cells during the cell cycle and is inactive in differentiated cells.
FIG. 7. Colocalization of cystatin B and cathepsin B by immunofluorescence confocal microscopy and electron microscopy analysis. Cell matrix (A, B); merging of the two signals (C); scatter plot (D). Pearsons correlation and overlap coefficient, calculated on 30 cells by LaserPix software, were 0.94 6 0.02 and 0.91 6 0.07, respectively. Electron microscope immunogold double labeling of cystatin B (small grains) and cathepsin B (large grains) (E). The relative position of the two signals is shown by the red circles. Bar, 0.2 mm. FIG. 8. Activity of cathepsin B in SHSY-5Y (AD) and cerebellar granule (EH) nondifferentiated (A, B, E, F) and differentiated (C, G, D, H) cells. The fluorescence images indicate the activity of cathepsin B, while those in phase contrast show the cell structure. 92 RICCIO ET AL.

It is surprising that cathepsin B and cystatin B are still bound to the cell matrix after the exhaustive salt extraction used in this procedure. An involvement of the nuclear matrix [34] in the selective activation of genes that express differentiated functions has been proposed by Robinson et al. [35]. These authors observed that the ovoalbumin gene, which is transcriptionally active in the chicken oviduct cells, is preferentially associated with the nuclear matrix, while the transcriptionally inactive b-globin gene is not associated. The matrix interaction of cystatin B and cathepsin B may imply a role of cathepsin B in the control of gene expression. The nuclear localization of cystatin B together with the cerebellar damage shown by Pennacchio et al. [9] in cystatin B knockout mice is consistent with a nuclear function of anti-protease during the differentiation of granule cells in the cerebellum.
The financial support of TelethonItaly (Grant E.711) is gratefully acknowledged. We are very grateful to Dr. Barry Howes for careful reading of the manuscript and to Dr. Angela Algeri for helpful suggestions.

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