Ferritin associates with marginal band microtubules

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Ferritin associates with marginal band microtubules Anthony A. Infante a , Dzintra Infante a , Muh-Chun Chan a , Poh-Choo How a , Waltraud Kutschera b , Irena Linhartová b,1 , Ernst W. Müllner c , Gerhard Wiche b , Friedrich Propst b,⁎ a

Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA Max F. Perutz Laboratories, Department of Molecular Cell Biology, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria c Max F. Perutz Laboratories, Department of Biochemistry, Medical University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

We characterized chicken erythrocyte and human platelet ferritin by biochemical studies

Received 16 September 2006

and immunofluorescence. Erythrocyte ferritin was found to be a homopolymer of H-ferritin

Revised version received

subunits, resistant to proteinase K digestion, heat stable, and contained iron. In mature

6 February 2007

chicken erythrocytes and human platelets, ferritin was localized at the marginal band, a

Accepted 8 February 2007

ring-shaped peripheral microtubule bundle, and displayed properties of bona fide

Available online 3 March 2007

microtubule-associated proteins such as tau. Red blood cell ferritin association with the

Keywords:

microtubules. During erythrocyte differentiation, ferritin co-localized with coalescing

Erythrocyte

microtubules during marginal band formation. In addition, ferritin was found in the

Platelet

nuclei of mature erythrocytes, but was not detectable in those of bone marrow erythrocyte

Ferritin

precursors. These results suggest that ferritin has a function in marginal band formation

Iron

and possibly in protection of the marginal band from damaging effects of reactive oxygen

Microtubule

species by sequestering iron in the mature erythrocyte. Moreover, our data suggest that

Marginal band

ferritin and syncolin, a previously identified erythrocyte microtubule-associated protein,

Cytoskeleton

are identical. Nuclear ferritin might contribute to transcriptional silencing or, alternatively,

Oxidative stress

constitute a ferritin reservoir.

marginal band was confirmed by temperature-induced disassembly–reassembly of

© 2007 Elsevier Inc. All rights reserved.

Introduction Three main components comprise the cytoskeleton of the avian erythrocyte and the cytoskeleton of nucleated erythrocytes in general: (1) a “membrane skeleton” consisting of spectrin filaments associated with actin and several other proteins, (2) a network of intermediate filaments composed primarily of vimentin extending from the nuclear envelope to the membrane, and (3) a ring of microtubules termed the

“marginal band” (MB) [1,2]. The MB is a characteristic component of the cytoskeleton of nucleated non-mammalian erythrocytes [3]. MBs consist of a stable bundle of microtubules that span the intracellular circumference of the cell, located directly underneath the plasma membrane, in the plane of flattening. During erythropoiesis, MB formation and its interaction with the membrane skeleton are responsible for the change in cell morphology from a spherical proerythroblast to a flattened, discoid-shaped mature erythrocyte [4]. MB

⁎ Corresponding author. Fax: +43 1 4277 52854. E-mail address: [email protected] (F. Propst). 1 Present address: Institute of Microbiology of the Academy of Sciences of the Czech Republic, Videnska 1083, CZ-142 20 Prague 4, Czech Republic. 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.02.021

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formation begins with the polymerization of tubulin alpha– beta dimers into long microtubules (several times the length of the cell diameter), a process nucleated by a microtubuleassembling complex at the centrosome [5]. As early as the second day of terminal erythropoiesis, an erythroid precursor contains between two and twenty microtubules extending from the centrosome in many random planes [6]. By the fourth day, these microtubules are bundled together in one plane and are aligned with the circumference of the cell. Microtubule bundling provides the force that is required to change the cell shape from a sphere to that of a flattened disc. Further studies showed that the MB continues to be relevant in the mature erythrocyte. Cells without MB collapse under physically stressful conditions (such as blood flow through capillaries, mimicked by passing cells through a needle), whereas cells with MB maintain their shape [4]. MBs are also important cytoskeletal elements in human and mouse platelets [7–10]. Platelets with defects in the MB display spherocytosis and an altered response to thrombin [8,9]. The formation and maintenance of the MB is believed to be accomplished through expression of microtubule-associated proteins (MAPs). In addition to tau, which is widely accepted as a MAP integral to microtubule polymerization in various contexts [11,12], characterization of the MB in various species has led to the identification of other traditional and nontraditional MAPs associated with this structure. Immunological studies support the association of MAP2 with newt erythrocyte MBs [13] but not with chicken erythrocyte MBs [14]. Syncolin, a globular, high molecular weight protein, which binds to microtubules in vitro, was also found to be associated with the MB of chicken erythrocytes [15]. In addition, other proteins such as F-actin and an ezrin-like protein have been shown to co-localize with tubulin in mature chicken erythrocyte MBs [16,17]. In search for novel MB-associated proteins we used an antiserum directed against purified MBs to isolate clones from a chicken erythroblast expression library. Unexpectedly, many of the clones identified with this antiserum encoded the protein ferritin which was not previously known for its association with microtubules. Ferritin is a ubiquitously expressed, highly conserved iron storage protein composed of tightly bound polypeptide subunits which surround a core of polynuclear iron [18,19]. In mammals, each ferritin molecule is composed of 24 subunits which belong to one of two highly related isoforms, one with molecular weight of about 19 kDa (L-chain) and the other of about 21 kDa (H-chain). Land H-chains associate at various ratios to form the 24subunit ferritin molecule of about 450 kDa which has the shape of a hollow ball, having an average outside diameter of 13 nm, and an average inside diameter of 7.8 nm. In addition to detoxifying a potentially harmful excess of low molecular weight iron compounds by storage, ferritin also reportedly serves as an “iron buffer”, limiting the supply of free iron in a labile-iron-pool. By regulating the labile-iron-pool, ferritin may help to regulate the redox state of a cell and to protect against the iron-catalyzed production of damaging reactive oxygen species (ROS) [20,21]. Ferritin has also been found in mitochondria [22,23] and in the nucleus [24,25] where it has been implicated in transcriptional regulation that may or may not require its direct binding to DNA [26,27].

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Here we characterized ferritin in chicken erythroid cells and show that the protein is associated with microtubules during MB formation in erythroblasts and is present in the MB and the nucleus of mature red blood cells (RBCs). In addition, we find ferritin associated with the MB in human platelets, indicating a possible general requirement of ferritin in this cytoskeletal structure.

Materials and methods Chicken organs and cells Chicken organs and mature RBCs for biochemical analyses were obtained from decapitated adult chickens. For immunofluorescence studies circulating blood cells (0.1 mL) were collected in phosphate buffered saline (PBS) on the day of the experiment from a live adult chicken or 10- to 18-day embryos. Bone marrow cells containing primarily RBCs at different stages of erythropoiesis were obtained by injecting PBS into the cavities of femurs from 18-day embryos. When the procedure required cells to be incubated for longer periods, the cells were kept in 25 mM glucose in PBS. Fresh human platelets were isolated from blood of healthy donors after obtaining informed consent, kept at 30 °C, and used on the same day.

Expression library screen and cDNA cloning and sequencing A chicken erythrocyte cDNA expression library in Escherichia coli strain Y1090 [28] was screened [29] using polyclonal antibodies raised in rabbits against the putative chicken erythrocyte MAP syncolin [15] or a polyclonal antiserum obtained from mice immunized with SDS–PAGE-purified syncolin. Positive clones were purified and analyzed by restriction analysis, Southern hybridization [29] and sequencing. The sequence determined for the full length coding region of chicken ferritin was deposited in GenBank® (accession number Y14698).

Ferritin purification Ferritin purification from RBCs and chicken organs was performed as described [30,31] with slight modifications. Briefly, tissues or packed RBCs were homogenized in 10 volumes of microtubule stabilizing buffer (MSB: 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8, 2.5 mM EGTA pH 8, 1 mM DTT, 0.1% Tween-20) using a tissuemizer for bursts of 10 to 20 s up to a total of 1 min. The lysate was centrifuged at 1600 × g for 5 min to remove large cellular debris, the supernatant was incubated for 10 min at 70 °C, followed by 10 min at 0 °C, and centrifuged at 16,500 × g for 20 min. The supernatant containing the heat-stable (70 °C) ferritin was layered onto a 10% sucrose cushion in MSB and centrifuged at 164,000 × g and 4 °C for 5 h. Supernatant, cushion, and pellet were collected. The pellet contained purified ferritin. Proteinase K treatment was performed with 20 μg/mL proteinase K (BRL, Rockville, MD), 0.2% SDS at 37 °C for 30 min [32]. Heat treatment for subunit dissociation consisted of heating the samples at 90 °C for 10 min in sample buffer (see below). Control horse spleen ferritin (HSF) was from Sigma (St. Louis, MO).

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Isolation of MB and nuclear fractions and protein analysis Erythrocyte cytoskeletons, MB components and nuclei were isolated as described [11,12]. Polyacrylamide gel electrophoresis (PAGE) under non-denaturing (5% gels) or denaturing conditions (15% gels) and immunoblot analysis was carried out as described [29]. Samples were loaded to yield approximately the same amount of ferritin. Detection of secondary antibodies on immunoblots was by ECL detection reagents (Amersham, Little Chalfont, UK). Iron content of proteins was determined by staining gels with 1% potassium ferrocyanide in 0.1 M HCl for 30 min [33]. The intensities of the protein bands (on Coomassie stained gels run in parallel) and iron bands were analyzed using the Un-Scan-It software (Silk Scientific, Oren, UT) to obtain iron/ protein ratios. Protein microsequencing of purified syncolin was carried out by Edman degradation as described [34].

Immunofluorescence and MB disassembly ex situ To preserve intact microtubules for immunocytochemistry chicken erythrocytes and human platelets were processed at 38 °C and 30 °C, respectively, as described [35]. To depolymerize the erythrocyte MB, after initial incubation at 38 °C, cells were incubated for 10 min at 25 °C, then 1 h at 0 °C. Cells at 0 °C were attached to cover slips, incubated for another hour at 0 °C, fixed with 3% paraformaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 for 1 min prior to staining [35]. To allow repolymerization of the MB after cold treatment, cells were transferred to room temperature for 10 min before re-incubating them at 38 °C for 30 min. Cells were then plated onto cover slips, incubated for 1 h at 38 °C, fixed, permeabilized and stained as described above. To follow the kinetics of MB repolymerization cells were incubated on cover slips at 0 °C for 90 min, then incubated for 1, 5, 15, 30, or 60 min at 38 °C prior to fixation. For quantification at each time point, a total of 200 cells were counted and the percentage of cells containing fully or partially formed MBs was determined. Image acquisition was carried out at 25 °C with a Zeiss Axiovert 100 confocal laser scanning microscope, Zeiss Apochromat 63× and 100× objectives with 1.40 numerical aperture, and a Zeiss LSM510 camera with LSM510 software. Images were arranged for figures using the Adobe Photoshop and Illustrator software with linear, if any, adjustments of brightness and contrast.

Antibodies A polyclonal rabbit anti-chicken ferritin antibody was obtained by immunizing rabbits (Cocalico Biologicals, PA) with chicken RBC ferritin purified as described above and subsequent additional preparative gel electrophoresis on a non-denaturing 5% polyacrylamide gel. The resulting antibody was used at a dilution of 1:100 in immunofluorescence and 1:500 on immunoblots. Other primary antibodies and their dilutions: monoclonal anti-α-tubulin (Biodesign International, Saco, ME), 1:200; monoclonal anti-β-tubulin (ICN Biomedicals, Aurora, OH), 1:200; polyclonal rabbit anti-HSF (Sigma), 1:100; affinity purified polyclonal goat anti-human ferritin (Y-16; Santa Cruz Biotechnology, Santa Cruz, CA), 1:50; monoclonal

anti-tau (TAU-1; Boehringer Mannheim, Germany), 1:200. Secondary antibodies: FITC-conjugated goat anti-mouse-IgG, 1:300; Texas Red-conjugated anti-rabbit IgG (Jackson, West Grove, PA), 1:100.

Results Characterization of ferritin in chicken erythrocytes In an attempt to obtain cDNA clones encoding syncolin, a putative novel MAP found to localize to MBs of chicken erythrocytes [15], we screened a cDNA expression library prepared from chicken erythroblast cells [28] using three different antisera. A first screen conducted with a rabbit polyclonal anti-syncolin serum [15] yielded 10 clones. A second screen, performed using the same serum after affinity purification against syncolin, yielded one additional clone. A further clone was obtained in a third screen where we used a polyclonal anti-serum that was obtained by immunizing mice with purified syncolin. The specificity of the rabbit antisera was shown previously [15] and was confirmed here, as was the specificity of the mouse serum (Fig. 1A). All sera specifically recognized the high molecular weight band presumed to represent the MAP syncolin on immunoblots of chicken erythrocyte protein extracts. Identical results were obtained in immunoblot analysis of chicken erythrocyte cytoskeleton preparations (unpublished results; Feick et al. [15]). From the total of 12 clones isolated, 1 clone obtained with the mouse serum and 4 clones obtained with the rabbit serum were partially sequenced and shown to encode the H-chain of chicken ferritin. Subsequently we determined the complete sequence which was deposited in the database (GenBank® accession number Y14698). The remaining seven clones also encoded the H-chain of chicken ferritin as revealed by restriction and hybridization analyses (not shown). Moreover, in parallel experiments, we determined the partial amino acid sequence of the putative syncolin protein band and found only sequences corresponding to ferritin (underlined in Fig. 1B). A comparison of the deduced amino acid sequence of our clones to that of bona fide chicken, horse, and human ferritin H-chains is shown in Fig. 1B. The sequence obtained from chicken erythrocytes is identical to the sequence obtained from the chicken genome which harbors only a single gene for the ferritin H-chain [36]. Furthermore, the chicken ferritin Hchain is highly related to the H-chains of horse and human (88.89% identity), and with 180 amino acids is almost identical in size. Since our result suggested novel features of erythrocyte ferritin, in particular that RBC ferritin was associated with microtubules of the MB, we decided to characterize the protein further. First, we purified ferritin from RBCs taking advantage of its heat stability and its large molecular mass [30,31]. Fig. 2A shows the non-denaturing PAGE analysis of proteins isolated from a number of chicken tissues using this procedure. A major band at an estimated Mr of 440 kDa was present in all tissues, including RBCs. All of the putative ferritins migrated to the same position on the gels and more slowly than horse spleen ferritin (HSF; see below Fig. 2E). This is consistent with previous reports and may be due in part to the high proportion

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Fig. 1 – Antisera specifically reacting with the putative high molecular weight MAP syncolin identify ferritin-encoding cDNA clones in expression library screens. (A) Chicken erythrocyte protein extracts were fractionated on 5% polyacrylamide gels and analyzed by immunoblotting using antisera obtained by immunization of rabbits or mice with purified syncolin. Both antisera detected a single high molecular weight band corresponding in size to the putative MAP syncolin [15]. (B) Sequence comparison of the cloned chicken erythrocyte ferritin H-chain (chicken ery; GenBank® accession number Y14698) to chicken ferritin H-chain genomic sequence (chicken gene; GenBank® accession number M16343) and the sequences of HSF (HSF; GenBank® accession number AB175616) and human ferritin (human; GenBank® accession number NM_002032) H-chains. Erythrocyte and genomic ferritin H-chains are identical, highly related to the H-chain sequences of horse and human (asterisks mark identities), and almost identical in size. Sequences obtained by peptide sequencing of purified high molecular weight syncolin are underlined.

of L-ferritin subunits (19 kDa) in HSF compared to chicken ferritin which contains only H-ferritin (21 kDa) [18] and/or to the less acidic isoelectric point of chicken ferritin (pI 6.6 compared to pI 4.9 for HSF [37]). Fig. 2A also shows that the 440 kDa band was resistant to proteinase K digestion. Multimeric ferritin dissociates into its subunits upon vigorous heating in SDS–PAGE conditions (90 °C for 8– 10 min). Fig. 2B shows that the high Mr band present under non-denaturing conditions was lost and its loss was complemented by the appearance of a single 21-kDa band. The 21-kDa subunit band was also evident upon heat denaturation of proteinase K-treated preparations (Fig. 2C). The resistance to digestion with proteinase K of both the 440-kDa band and the 21-kDa subunits is a well-established characteristic of ferritin [32]. A major property of ferritin is that it contains iron. Fig. 2D shows that the 440-kDa band derived from all the tissues examined contained iron. Comparison of the relative iron content per unit ferritin indicated that spleen and to a lesser degree liver, organs which are known to store iron, had the highest iron to ferritin ratio (1.9 and 1.2, respectively, in

arbitrary units). RBC ferritin and heart ferritin contained less iron, but at a level comparable to that of liver (0.9 in arbitrary units). Thus, chicken RBC ferritin is similar to ferritin from other chicken tissues in terms of its size, subunit composition, protease resistance and iron content. Moreover, liver and RBC ferritin exhibited the same isoelectric point (6.3–6.5; data not shown). To confirm the identity of the putative RBC ferritin, we carried out immunoblots using anti-HSF antibodies. This antibody reacted with both the putative intact ferritin and ferritin subunits (Fig. 2E). In addition, we prepared and used antibodies against RBC ferritin. In all of the following immunological examinations anti-chicken RBC ferritin antibodies and the anti-HSF antibody gave the same results.

Ferritin is associated with the MB and is present in the nucleus As a first step towards elucidating ferritin function in chicken RBCs, we decided to study its subcellular localization. We performed a double-labeling immunofluorescence analysis using anti-ferritin and anti-α-tubulin antibodies (Fig. 3).

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Fig. 2 – Biochemical properties of chicken RBC ferritin. Lysates of the indicated chicken tissues were subjected to ferritin-enrichment extraction and analyzed by PAGE. (A) Non-denaturing 5% gels display a common high molecular weight band derived from all tissues (lanes labeled − pK) which is resistant to proteinase K digestion (lanes labeled +pK). (B) Heat treatment of tissue extracts at 90 °C for 10 min in sample buffer (lanes labeled +H) led to loss of the high Mr band (arrowheads; lanes labeled − H) and resulted in a prominent band at approximately 21 kDa (asterisks). (C) Tissue extracts were left untreated (−pK lanes) or digested with proteinase K (+pK lanes), heated and analyzed on denaturing gels. The 21-kDa band (asterisk) is resistant to proteinase K. (D) Detection of iron in the high Mr band; top panel stained for protein, bottom panel stained for iron. (E) Reaction of anti-HSF anti-serum with the high Mr band on immunoblots of non-denaturing gels (top panel) and the 21-kDa band on denaturing gels (bottom panel – asterisk). Ferritin-enriched chicken tissue extracts were run along with HSF. The position of HSF subunit on the denaturing gel is obscured by its exceptionally strong reactivity with the antibody. The positions of Mr values indicated were determined from standards.

Ferritin (Figs. 3A, D) was found in the nucleus and a ringshaped structure at the periphery. Tubulin was present in the MB at the periphery of the cells (Figs. 3B, E). The merge of the ferritin and tubulin signals (Figs. 3C, F; yellow staining) indicated co-localization of ferritin and tubulin at the MB. The same results were obtained when anti-β-tubulin antibodies were used to detect tubulin (not shown). To rule out that under the conditions applied antibodies would decorate the MB in a non-specific way, we used anti-vimentin antibodies under the same conditions. In accordance with published reports [1] we detected vimentin in the perinuclear cytoskeleton with tufts of filaments extending towards the plasma membrane, but not in the nucleus, or at the MB, demonstrating the specificity of the analysis (not shown). To test whether MB localization of ferritin is restricted to chicken erythrocytes or might be of broader relevance, we analyzed human platelets which have been demonstrated to contain MBs [38] using an antibody capable of detecting human ferritin in fixed cells (anti-human ferritin Y-16 [39]). By co-staining for tubulin we

found that ferritin in human platelets is indeed associated with the MB (Fig. 3G–I) suggesting that ferritin localization at the MB is a general feature of MB containing blood cells. To further substantiate the results obtained by immunocytochemistry we prepared subcellular fractions of chicken RBCs and analyzed them by immunoblot using anti-HSF and anti-tubulin antibodies (Fig. 4). When cells were lysed under conditions which rendered the MB intact, ferritin was found in the soluble fraction and in insoluble material containing the cytoskeleton and nuclei. As expected, under these conditions tubulin was only found in the insoluble cytoskeleton fraction. Treatment of this fraction with colchicine led to disintegration of the MB and solubilization of tubulin and MB-associated proteins. As a result, following centrifugation, tubulin was now found in the supernatant, along with ferritin, confirming the immunocytochemical analysis which had shown colocalization of tubulin and ferritin in the MB. The remaining pellet contained nuclei and insoluble components of the RBC cytoskeleton. Tubulin was absent or present only in trace

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Fig. 3 – Immunolocalization of ferritin and tubulin in chicken erythrocytes and human platelets. Double immunofluorescence analysis of RBCs with anti-chicken erythrocyte ferritin (A) or anti-HSF (D) and anti-α-tubulin (B and E) antibodies. Ferritin was found in the nucleus (arrowheads) and at a ring-shaped structure in the cell periphery (A and D; arrows). Tubulin was present in the MB (B and E; arrows). The merge of the two signals (C and F; yellow) showed co-localization of ferritin and tubulin in the MB (arrows) but only ferritin was detected in the nucleus (arrowheads). Likewise, human platelets were analyzed by double immunofluorescence analysis with anti-human ferritin (G–I) and anti-α-tubulin (J–L). The merge of the two signals is shown in yellow (M–O). Ferritin was found in the cytoplasm, but also associated with the MB (arrows).

amounts in this fraction, but ferritin was still present (Fig. 4), consistent with the immunocytochemical localization of ferritin in the nucleus (Fig. 3).

Ferritin localization during MB formation

Fig. 4 – Subcellular localization of ferritin in erythrocytes by biochemical fractionation. Liver ferritin and tubulin were used as positive controls for reactions with anti-HSF antibody (HSF) and anti-α-tubulin antibody (α-Tub). Cytoplasmic fractions 1 and 2 were supernatants (soluble fractions) collected from two cycles of cell lysis [12]. The cytoskeleton fraction was the pellet resulting from these centrifugations and contained the MB and nuclei. Treatment of this fraction with colchicine followed by centrifugation yielded the MB fraction which contained disaggregated MB tubulin and associated proteins in the supernatant (MB), and the nuclear fraction (nucleus) in the pellet. Samples were denatured and run on 5% gels.

Having shown that ferritin is localized in the MB of mature erythrocytes using both immunohistochemistry and biochemical techniques, we wanted to determine the timing of ferritin association with the MB. We first analyzed the localization of ferritin during the process of MB formation during erythroblast differentiation in vivo. Bone marrow erythroid progenitors at various stages of maturation were collected, fixed, and labeled with anti-chicken RBC-ferritin antibodies and anti-tubulin antibodies (Fig. 5). The results indicate that ferritin and tubulin co-localize during the process of MB formation in vivo. Ferritin co-localized with unbundled, centrosomal microtubules in early erythroblasts (Fig. 5, row A, arrows). This co-localization persisted as the microtubules were becoming bundled to form the MB in basophilic (Fig. 5, rows B and C, arrowheads) and polychromatophilic erythroblasts (Fig. 5, row D), as well as in reticulocytes (Fig. 5, row E), whose MB is comparable to that of mature erythrocytes. Thus, ferritin was found to be

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Fig. 5 – Ferritin association with microtubules during erythroid differentiation in vivo. Double-labeling immunofluorescence analysis of differentiating bone marrow erythroid progenitors was performed with anti-α-tubulin and anti-chicken ferritin antibodies as indicated. Merges of the images are seen in the right column. Row A: early erythroblast with centrosomal microtubules (arrows). Rows B and C: basophilic erythroblasts with bundling microtubules (arrowheads). Row D: polychromatophilic erythroblast with bundled microtubules. Row E: reticulocyte with discoidal MB similar to that of erythrocytes obtained from adult chickens. All images were taken with the confocal microscope. Bar = 5 μm.

associated with microtubules during all stages of terminal erythropoiesis. On the other hand, nuclear localization of ferritin was not detected in immature erythrocytes. A second method used to test if ferritin might be required for MB formation involved observing ferritin localization during ex situ MB formation. MBs of mature erythrocytes

were depolymerized by incubation at 0 °C and then repolymerized at 38 °C. The cells were stained for ferritin, tubulin (both alpha and beta) and the MAP tau to be able to compare the behavior of ferritin with that of a well characterized MAP (Fig. 6). At 38 °C, ferritin, tubulin, and tau were detected in the MB (arrows). These signals disappeared in cells that were fixed

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after incubation at 0 °C, but appeared again when the cells were re-incubated at 38 °C, demonstrating that ferritin behaved like a bona fide MAP. Ferritin staining in the nucleus was observed at both temperatures (arrowheads). Similar results were obtained when colchicine was used as a microtubule depolymerizing reagent and the MB reformed after colchicine wash out (data not shown). Finally, we determined the detailed kinetics of the behavior of ferritin in ex situ MB formation. Following depolymerization at 0 °C, MB structures were not detected among cells that were not re-incubated at 38 °C (0 min of re-incubation; Fig. 7). After re-incubation at 38 °C for 1 min, about 9.0% of the cells contained partial or fully formed MBs as revealed by tubulin staining. There was no significant increase in the proportion of MB containing cells after 5 min (9.5%). However, after 30 min and 60 min of re-incubation, there were large increases in the proportion of MB containing cells, reaching 37% and 47%, respectively. Merged images indicated co-localization of ferritin and tubulin at partial and complete MB structures. Some cells contained short MB fragments along the flatter plane of

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the disc, forming structures resembling parentheses or brackets staining with anti-tubulin and anti-ferritin antibodies (Fig. 8, arrows). Other cells contained longer, “U”-shaped incomplete or almost fully formed MBs (e.g. Fig. 8, at 60 min). In agreement with published findings [40] these observations suggest that MB formation is initiated at two different loci in the cell. The two MB fragments ultimately meet at the cusps of the disc at both ends of the cell to form the final MB structure. These results on the kinetics of MB formation ex situ again demonstrated that, similar to in vivo MB formation, ferritin is localized with tubulin in the MB as it forms.

Discussion Biochemical characteristics of chicken RBC ferritin In search for novel proteins associated with the MB of mature chicken erythrocytes we discovered that ferritin is present throughout erythroblast differentiation, is associated with

Fig. 6 – Microtubule-dependent localization of ferritin at the MB. At 38 °C, the MB (arrows) was stained by anti-α-tubulin (α-Tub), anti-β-tubulin (β-Tub), anti-ferritin (HSF), and anti-tau (Tau-1) antibodies as indicated. Signals from the MB were not observed in cells exposed to 0 °C for 90 min. Signals reappeared when the MB was allowed to form again during renewed incubation at 38 °C for 90 min (0 °C to 38 °C). Ferritin staining is observed in the nucleus at both temperatures (arrowheads). Bar = 10 μm.

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Fig. 7 – Ferritin localization during ex situ formation of the MB. Cells pretreated at 0 °C for 90 min were reincubated at 38 °C for the indicated time periods to allow MBs to repolymerize. Cells were double stained with anti-α-tubulin (α-Tubulin) and anti-chicken-ferritin (Ferritin) antibodies. The right column shows the merge of the two images (Merge). Fully and partially formed MBs were seen at all times, except after 0 min of repolymerization. There was an increased proportion of cells with fully or partially formed MBs as repolymerization time was increased. Bar = 50 μm.

microtubules during MB formation, and is localized at the MB of circulating RBCs. Biochemical tests showed that chicken RBC ferritin is similar to ferritin found in several chicken organs in size, presence of iron, resistance to proteinase K, reactivity with anti-HSF antibodies, and in that the erythrocyte ferritin complex could be dissociated to produce a single ∼ 21 kDa ferritin subunit band (Fig. 2). These results are consistent with previous reports indicating the presence of ferritin in erythrocytes, including those of mammals which lack the nucleus and the MB [19,33,41–43]. Avian ferritin is composed solely of H-ferritin subunits [36,37,44]. This is also

the case in plants and bacteria which only contain H-subunit homopolymers [45]. In contrast, mammalian cells express heteropolymers of ferritin consisting of H- and L-subunits. Moreover, the H:L-ferritin ratio is tissue-specific [46], implying that tissue-specific differences in ferritin function are accommodated for by varying the subunit composition. Nevertheless, the H-ferritin subunit is essential in mammals. Mice deficient in H-ferritin but retaining intact L-subunits fail to develop to term [47]. The fact that chicken ferritin consists solely of H-ferritin homopolymers, indicates that the chicken H-subunit is sufficient to perform adequately in iron storage

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Fig. 8 – Presence of ferritin on partial MB fragments during ex situ MB formation. Cells were fixed at various time points during temperature induced re-polymerization of microtubules as in Fig. 7. Arrows point to the forming MB which stains with both anti-chicken-ferritin and anti-tubulin antibodies. Bar = 5 μm.

and sequestration and in whichever additional functions it might have in erythrocytes.

Erythrocyte ferritin and MB formation The results presented here suggest that ferritin may have a novel role in chicken RBCs acting as a component of the MB. Immunofluorescence studies demonstrated the association of ferritin with the MB of mature erythrocytes. Both temperature- and colchicine-induced destruction of the MB led to the concomitant release of ferritin and tubulin from the peripheral ring structure. Furthermore, when cells were allowed to re-polymerize their MB, ferritin was found to associate with nascent MB fragments (Figs. 5, 6, and 7). In this, ferritin displayed properties similar to tau, a bona fide MB-associated MAP [12,14]. These results indicate that ferritin is indeed associated with the MB and not merely with the membrane skeleton at the periphery of the cell [1]. If that were the case,

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destruction of the MB would not have led to release of ferritin into the cytoplasm, and the oval ring-shaped ferritin signal at the periphery of the cell would have been retained despite depolymerization of microtubules. In a separate set of experiments, we analyzed the intracellular localization of ferritin at different stages of erythroid progenitor differentiation. Cells at all stages of microtubule polymerization and bundling during MB formation were obtained from bone marrow. We could show that ferritin associated with microtubules coalescing into the MB during in vivo erythroid maturation (Fig. 5). These results are of particular interest in light of the recent finding that HSF can directly interact with, and bind to microtubules [48,49]. Whereas these studies by Hasan et al. [48,49] provide evidence for a role of microtubules in iron metabolism, the work presented here underlines a complementary aspect of ferritin microtubule interaction: our results of both in vivo MB formation and temperature-induced de- and repolymerization of the MB are consistent with a possible role of ferritin in microtubule rearrangement and MB formation. Alternatively, as discussed below, association of RBC ferritin with the MB might not be involved in its formation but pertain to a specific function of ferritin in erythrocytes and human platelets. The observed association of ferritin with microtubules raises the question whether ferritin contains a microtubule binding domain. Analysis of the primary amino acid sequence did not reveal prominent stretches of basic amino acids which are found in microtubule-binding domains of classical MAPs, nor was there significant homology to any of these MAPs which would allow to delineate a potential microtubule binding domain. Consistent with our own observations and with results obtained by Hasan et al. [48], it is conceivable that ferritin binds to microtubules only in its multimeric form. Complex formation might create additional surfaces in the multimeric protein which could permit its interaction with microtubules. On the other hand, isoforms of ferritin might exist that contain or lack a microtubule binding domain. Isoforms containing a microtubule-binding domain could be specifically expressed in cell types containing an MB, but not in other cell types. This does not seem likely in chicken where only one isoform encoded by a single H-chain gene has been detected. However, in humans, the situation is more complex since at least two subunits (H and L) exist. This raises the possibility that certain cells express multimeric ferritin complexes containing H- and L-subunits at a unique ratio which may facilitate microtubule binding. Also, evidence for the existence of ferritin H-chain isoforms that differ slightly in amino acid sequence has been reported [22,50]. It remains to be seen if ferritin expressed for example in human platelets which contain an MB is composed of unique subunits that differ in sequence and microtubule binding activity from ferritin found in cells which lack an MB.

Evidence for identity of ferritin and the MAP syncolin Syncolin was originally presumed to be a novel high molecular weight MAP specifically expressed in chicken RBCs, because it co-purified with RBC tubulin, bound to microtubules in vitro,

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and localized to the MB [15]. In order to obtain cDNA clones encoding this protein, syncolin-specific antisera were raised, affinity purified, and used to screen a chicken erythroblast cDNA library. A total of 12 cDNA clones were obtained. To our surprise, sequencing of these clones revealed that they all encoded authentic chicken H-ferritin. Thus, the question arose whether syncolin was in fact a polymeric high molecular weight form of ferritin. Apart from the cloning of ferritin cDNA clones with syncolin-specific antibodies, several additional findings support this view. First, affinity-purified anti-syncolin antibodies cross-reacted with ferritin, and anti-ferritin antibodies reacted with purified syncolin on immunoblots. Secondly, syncolin and the polymeric high molecular weight form of ferritin displayed the same molecular weight on polyacrylamide gels (not shown). Third, partial amino acid sequence determination by microsequencing of highly purified syncolin protein yielded only ferritin sequences. Furthermore, ferritin behaved like syncolin in temperaturedependent de- and re-polymerization of the MB (Figs. 6 and 7) [15]. And finally, electron microscopic analysis of purified syncolin revealed a shape of the molecule [15,51] that was very similar to the 13-nm hollow ball structure reported for ferritin [52]. Taken together, these findings provide strong evidence that syncolin is indeed RBC ferritin.

Ferritin in the nucleus of avian erythrocytes The immunofluorescence studies presented here revealed a distinct localization of ferritin not only at the MB, but also in the nucleus of circulating RBCs. In erythroid cells isolated from bone marrow, nuclear staining was not apparent, most likely indicating a translocation of ferritin into the nucleus at late stages of erythroid maturation. Ferritin has been found in nuclei of other cell types as well and has been shown to bind to DNA [53]. The function of nuclear ferritin is currently under debate. The protein has been implicated in protection of DNA in the nuclei of avian corneal epithelial cells and astrocytoma cells [24,54,55]. Although mature erythrocytes loaded with hemoglobin bound oxygen might be under considerable mutagenic stress, their DNA is transcriptionally silent and DNA damage should be without consequences. There are, however, other propositions for the role of ferritin in the nucleus. Nuclear ferritin has been implicated in the regulation of β-globin gene expression in human K562 cells [26,27]. In addition, increased expression of the H-subunit of mouse ferritin corresponded with decreased levels of β-globin mRNA in mouse erythroleukemia cells [56]. Thus, it is conceivable that ferritin has a transcription regulatory function and might contribute to transcriptional silencing during erythrocyte differentiation. Another possibility is that the nucleus serves as a reservoir for any ferritin not ultimately associated with and performing its function at the MB. In most cell types, the synthesis of ferritin is regulated at the translational and/or transcriptional level in response to fluctuations in iron levels and other stresses, especially oxidative and inflammatory conditions [57–59]. Since circulating avian erythrocytes do not have the capability to transcribe DNA and are devoid of RNA they might have utilized another way to regulate cytoplasmic or MB-associated ferritin levels. Mechanisms of transporting ferritin into the nucleus have been described [25,60] and it

remains to be seen whether such mechanisms function in erythrocytes.

Ferritin and cytoprotection in erythrocytes and platelets Ferritin is traditionally considered an iron-storage protein and might well perform this function in erythrocytes as well. During differentiation, erythroblasts synthesize hemoglobin and require a vast amount of iron obtained from a putative free iron pool that is possibly also modulated by a ferritin–iron buffer system [56,61]. However, non-traditional functions of ferritin have been suggested as well [59]. For example, ferritin has been implicated in signal transduction [21,58,62], prohormone cleavage in the serum [63], and transport of prions across the intestinal epithelium [64]. As discussed above, our findings also provide evidence for a novel role of ferritin in MB formation and support the possibility of a nuclear function. Owing to its iron sequestering activity, ferritin might also function as a cytoprotectant against iron-induced oxidative stress in mature RBCs and platelets. In diverse systems, ferritin expression is enhanced by pro-oxidant conditions such as heat shock [57,65], excess iron [57], UV-A irradiation [66], and inflammation [67]. Thus, ferritin may have a major function as a defense against stress by serving as a cytoprotectant against oxidants [59]. This has been demonstrated in endothelial cells and HeLa cells [68,69]. The high level of iron in RBCs and platelets [70] makes their proteins particularly vulnerable to oxidative damage, which in contrast to other cells can be neither compensated by proteasomal turnover nor by de novo re-synthesis. Although the lifetime of RBCs and platelets is short, the maintenance of their structure and function might depend on minimizing the effects of ROS. Oxidative stress has recently been shown to result in depolymerization of microtubules [71]. This would have deleterious effects on erythrocytes and platelets whose characteristic shape is absolutely dependent on an intact MB [4,7,9,10]. Damage to the cytoskeleton leading to changes in the flattened shape of nucleated RBCs might compromise oxygen supply through blood capillaries and/or would lead to an increased rate of hemolysis. The latter has been observed in several forms of spherocytosis, where other components of the cytoskeleton (e.g. ankyrin or spectrin) are damaged [72]. Likewise, defects in the MB of platelets have been associated with spherocytosis, as well as with defects in the response of platelets to thrombin, and with certain human bleeding syndromes [8,9]. Our finding that both in RBCs and in platelets that ferritin is associated with the MB suggesting that it offers protection from ROS-induced damage to cytoskeletal structures.

Acknowledgments We are grateful to F. Lottspeich for protein microsequencing, to G. Gilarde and I. Fischer for help with confocal microscopy and to W. Clebowicz for assistance with tissue collection. This research was supported by a Wesleyan Project Grant to A.I., by funds from the Howard Hughes Medical Institute Undergraduate Science Education Program to Wesleyan University, a grant from the Austrian Federal Ministry of Science and

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Transport to G.W., and grants from the Austrian Science Fund to F.P. (Project No. SFB F607), E.W.M. (Project No. SFB F2809), and G.W. (Project No. SFB F011).

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