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Cell Biology International (2008) 32, 121–127 (Printed in Great Britain)
Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells
Jing‑Jing Huang1, Hong‑Li Yan1, Yuan‑jian Gao, Shu‑Han Sun*, Yan He, Fei‑Xiang Ding, Qian Mei and Geng Xue
Institute of Molecular Genetics and Engineering, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China


Abstract

Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host-derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP–annexin B1 plasmid and transfected it into SiHa cells. We found that GFP–annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12-myristate 13-acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.


Keywords: Annexin B1, Calcium, Immunohistochemistry, Protein kinase C.

1These authors contributed equally to this work.

*Corresponding author. Present address: Department of Medical Genetics, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China. Tel./fax: +86 21 2507 0331.


1 Introduction

Annexins are Ca2+-dependent phospholipid-binding proteins that belong to an evolutionarily-conserved multigene family, the members of which are expressed throughout the animal and plant kingdoms. These mainly intracellular proteins are localized either free in the cytosol or associated with cellular membranes or cytoskeletal proteins (Gerke and Moss, 2002). Despite the fact that these proteins lack secretary signals, many previous reports have documented their presence in the extracellular milieu. Annexin A1 was detected by immunoblotting analysis in the supernatants of colonic biopsies incubated in culture media, and in the luminal colonic perfusates of ulcerative colitis (UC) patients (Vergnolle et al., 2004). Annexin A6 was shown to be translocated from an intracellular pool to the external basolateral surface of the mouse mammary-duct epithelium upon the onset of lactation (Rocha et al., 1990). Moreover, annexin A2 showed a similar pattern of expression on endothelial cell surfaces (Hajjar et al., 1994).

Nevertheless, annexins are not secreted through the classical exocytic pathway, and the lack of a hydrophobic leader peptide prevents them from being targeted to the endoplasmic reticulum. Experiments in many cell lines have shown a direct correlation between the protein kinase C (PKC) phosphorylation and annexins' calcium-dependent membrane binding or exocytosis (Creutz et al., 1978; Liu et al., 1996; Gerke and Moss, 1997; Chander et al., 2001).

Taenia solium metacestode infection is a zoonotic disease with a wide distribution throughout the world. It presents a serious threat to human health by causing neurocysticercosis, and is responsible for important economic losses in pig breeding, especially in developing countries (Garcia and Brutto, 2000). In order to obtain the protective antigens required to develop nucleic-acid vaccines against Cysticercus cellulosae, an expression library was screened using sera from infected humans and/or pigs (Sambrook et al., 1989). One clone, designated as cC1, was recognized by sera from both humans and pigs. Sequence analysis revealed that it had typical carboxy (C)-terminus and distinct amino (N)-terminus structures. The cDNA encoded a novel member annexins family, which was designated as annexin B1 (Yan et al., 2002).

Annexin B1 was isolated from a Cysticercus cellulosae library by immunological screening, implying that it might be able to be secreted into the extracellular milieu. It was in this context that we investigated whether annexin B1 was in fact secreted, and if this is true, what is the mechanism underlying the secretion. We firstly isolated Cysticercus cellulosae and peripheral muscles from infected pigs and examined the histological distribution of annexin B1 by using immunohistochemical analysis. Then we constructed a pEGFP–annexin B1 plasmid and stimulated the pEGFP–annexin B1 transfected SiHa cells with both phorbol 12-myristate 13-acetate (PMA) and ionomycin.

2 Material and methods

2.1 Samples

We obtained permission for performing the research protocols and all experiments, following the guidelines of the ethics committee of the Second Military Medical University (SMMU). Muscle samples from five condemned pigs naturally infected with the larvae of T. solium were obtained from Experimental Animal Centre of Hebei province. These pigs were diagnosed according to the national standard and confirmed at slaughter. Samples, including one or more larvae and surrounding muscle tissues, were selected from heavily infected skeletal muscles. A total of 32 lesions were divided in two groups randomly: some were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5μm sections, and then stained with hematoxylin–eosin; the others were fixed for immunohistochemical staining. Cystic fluid of T. solium cysticerci was obtained by using the following method. Intact cysticerci were separated with great care and cystic fluid was collected with a fine needle penetrating into the tegument. Blood samples were collected from each pig and kept at 4°C overnight and then the serum was separated and stored at −20°C. In addition, two T. solium cysticercosis-free pigs (negative diagnosis and without any history of cysticercosis) were also obtained and their muscle and serum samples were collected. Serum samples were presented by Dr. Wang and they were from patients with cysticercosis (n=3), toxocariasis (n=2), schistosomiasis (n=3) and hydatidosis (n=2), all proven clinical and laboratory diagnosis.

2.2 Detection of annexin B1 by double antibodies sandwich ELISA (Ag-ELISA)

We have previously obtained a monoclonal antibody against annexin B1 (2B10H5) with high sensitivity and specificity (Gao et al., 2007). The serum samples were tested in duplicate for the detection of annexin B1 using a sandwich ELISA (Ag-ELISA) as described before (Dorny et al., 2000, Pouedet et al., 2002). Ninety-six-well plates (Sino-American Biotechnology Company) were coated with 1μg MoAb 2B10H5, per well diluted in carbonate buffer (1:100 per well, pH 9.6) overnight at 4°C. Plates were washed three times for 5min with PBS contain 0.05% Tween 20 and blocked with 100μl PBS containing 1% BSA for 2h at 37°C before washing again. The serum samples were treated with 5% TCA and incubated for 20min at room temperature. After centrifugation, the supernatant was neutralized using a 0.6M sodium carbonate/bicarbonate buffer (pH 10.0). Serum samples diluted 1:50 in PBS plus 1% BSA were added and incubated for 1h at 37°C.

Plates were washed again followed by the addition of sheep anti-pig IgG coupled to horseradish peroxidase (HRP, Jingmei Biotech), which was developed with 3,3′-diaminobenzidine (DAB) substrate. Plates were washed three times after each reaction step. Optical density readings at 450nm were carried out in an ELISA processor. Results were expressed as optical density indices (ODI). An ODI ≥2.1 was considered positive.

2.3 Immunohistochemistry

The small pieces of tissue were fixed in acetone at 4°C and embedded into paraffin blocks. Tissue blocks were sectioned at 5μm and mounted on poly-l-lysine-coated slides. Antigen retrieval was performed by boiling sections in 10 mM citrate buffer (pH 6.0) for 10min and endogenous peroxidase activity was quenched with methanol containing 0.3% hydrogen peroxide for 15min. Non-specific binding was blocked with 5% normal goat serum in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 20min. Slides were incubated in 1:50 dilution of MoAb antibody 2B10H5 (prepared by our laboratory) in 1% BSA in PBS at 4°C overnight. Slides were washed and incubated in goat anti-mouse IgG (Vector, USA) diluted 1:100 with 1% BSA in PBS for 60min at room temperature. Detection of annexin B1 was performed by incubation of sections for 10min with 0.2mg/ml Metal-3,3′-diaminobenzidine (DAB) (Sigma, USA) in Tris–HCl buffer pH 7.6, and counterstained with hematoxylin. Control sections were incubated with non-immune mouse serum at the same dilution. Sections were examined in an Olympus microscope.

2.4 Plasmid construction

The full-length cDNA of annexin B1 and human annexin A5 was originally cloned into the pJLA503 vector (Zhang et al., 2004). The resultant constructions, designated as pJLA503–annexin B1 and pJLA503–annexin A5, were used as template DNA for PCR. The oligonucleotide primers designed to amplify the coding region of annexin B1 were forward primer: 5′-CGCCTACTGTCGCTCCCTGGTTC-3′ and reverse primer: 5′-GCTATTATGCAGGGCCGATGAGTTTCAAG-3′. Full-length human annexin A5 cDNA was amplified by PCR using forward primer 5′-GGCCCAGCTAGCTTCAAAATGTCTACTG-3′ and reverse primer 5′-ACCATTTGTCGACGCTCAGGCCGTGT-3′ based on the cDNA sequence of annexin A5. The eukaryotic expression recombinant plasmids, pEGFP–annexin B1 and pEGFP–annexin A5, were constructed by inserting the amplified fragment into the EcoRI and SalI sites of pEGFP–N1 (Clontech, USA) and transformed into Escherichia coli DH5α. The transformants were confirmed by DNA sequencing.

2.5 Cell culture, transfection and treatment

SiHa cells were grown to 90% confluence at 37°C and 5% CO2 in air in DMEM (Sigma, USA), supplemented with 10% FCS (Life Technologies, USA). The recombinant plasmids, pEGFP–annexin B1, pEGFP–annexin A5, and empty vector pEGFP–N1 were transfected into SiHa cells using the lipofectAMINE and PLUS Reagent (Invitrogen, USA) according to the protocol provided by the manufacturer. At 24h after transfection, cells were harvested from dishes and grown overnight on thin coverslips in 12-well plates. After being washed in PBS, cells were incubated in serum-free medium for 24h. Subsequently some of the cultures were exposed to 20nM phorbol ester PMA (Sigma) in Ringer's buffer(145mM NaCl, 4.5mM KCl, 1mM MgCl, 2mM CaCl, 5mM Hepes and 10mM glucose, pH 7.4) for 15, 30 and 45min, respectively. Another set of cultures were treated by 10μM ionomycin (Sigma) in the same buffer for different periods of time (10, 30 and 60s). The treated cells were immediately fixed with 3.7% formaldehyde. We analyzed the fluorescence intensity in the nucleus and plasma by using Leica software 2.61. The fluorescence intensity ratio Rn of nucleus vs. plasma was calculated as follows:where Fn is fluorescence intensity of the nucleus; An is the area of the nucleus; Ft is fluorescence intensity of the whole cell; and At is the area of the whole cell.

2.6 Western blotting

After being transfected for 48h, cells were dissolved in RIPA buffer containing a protease-inhibitor cocktail (Sigma). Protein concentrations were determined using a MicroBCA kit (Pierce, USA). Total cell protein (30μg) was loaded on a 10% SDS–polyacrylamide gel (Bio-Rad, USA) and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk in 0.05% Tween/PBS (PBS-T), membranes were washed 3 times with PBS-T for 10min. To detected the two proteins, the membranes were incubated with monoclonal anti-annexin B1 (MoAb 2B10H5) diluted 1:500 and monoclonal anti-human annexin A5 (Sigma) diluted 1:1000 for 4h, followed by washing as described above. The membranes were incubated with a 1:1000 dilution of HRP-conjugated goat anti-mouse IgG (Sigma) as second antibody, washed with the same procedure above, and revealed with DAB substrate (Amersham, Sweden).

2.7 Measurement of intracellular calcium in cells after treatment with PMA and ionophore

The transfected SiHa cells were grown overnight in glass bottom dishes (Willco Wells, USA). After being starved for 24h, cells were loaded for 30min with 5μM acetoxymethylester of the fluorescent calcium indicator Rhod-2 (Molecular Probes, USA) in Ringer's buffer at 37°C. The cells were washed 3 times in Ringer's buffer, and the intracellular calcium concentration was measured using confocal laser microscope (Leica, GER). Rhod-2 fluorescence was excited at 540nm, with emission monitored through a 605nm barrier filter.

2.8 Statistical analysis

Data are expressed as mean±S.E. and the number of experiments is shown as n. Statistical comparisons were made with the use of Student's t-test. A value of P<0.05 was regarded as significant.

3 Results

3.1 Histological distribution of annexin B1

We first determined whether annexin B1 could be found in the cystic fluid of T. solium cysticerci and the sera of infected humans/pigs. Double-antibody sandwich ELISA demonstrated the following scores: 19 out of 27 (70%) in the cystic fluid of T. solium cysticerci; 4 out of 5 (80%) in the sera of pigs with cysticercosis; and 2 out of 3 (67%) in human sera with cysticercosis. No antigens were detected in the sera of patients with the helminth infections mentioned above or in cysticercosis-free pigs.

We further detected the histological distribution of annexin B1 in the T. solium cysticerci. Strong brown signals were mainly detected in the bladder wall of the T. solium cysticerci and the layer derived from the host. Intriguingly, signals were only present in the inflammatory layer consisted of macrophages that transformed into epithelioid cells (Fig. 1), but not in the fibrous layer mainly consists of mesenchymal cells. These data suggested that annexin B1 was able to be present out of the parasite and might be related to inflammatory reaction.


Fig. 1

Histological distribution of annexin B1. The procedure of immunohistochemical staining is described in Section 2. (A) Immunohistochemical staining. (B) hematoxylin–eosin staining. Strong brown signals were mainly detected in the tegument of Cysticercus cellulosae (arrowhead) and host-derived bladder wall (thick arrow) (A). The inflammatory layer consists of macrophages that transformed into epithelioid cells (arrowhead); the fibrous layer mainly consists of mesenchymal cells (thick arrow) (B). H, host; C.c, Cysticercus cellulosae; I.L, inflammatory layer; F.L, fibrous layer. Magnification of photos: 100×.


3.2 Identification of annexins in transfected SiHa cells

Western blot analysis was used to determine whether these GFP chimeras were expressed as stable and intact proteins. Fig. 2 shows that GFP–annexin B1 and GFP–annexin A5 were indeed present in these cells. The molecular masses of the antigens corresponded to those reported in the literature (those being GFP–annexin B1, 65kDa; GFP–annexin A5, 64kDa).


Fig. 2

SDS–PAGE of total lysates of transfected SiHa cells and Western blotting of GFP–annexin B1 and GFP–annexin A5. (A) Protein markers were loaded in lane 1; total cellular lysates from pEGFP–annexin B1 transfected cells was loaded in lane 2; the lysates from pEGFP–annexin A5 transfected cells were loaded in lane 3. (B) Immunoblot analysis of GFP–annexin B1 (lane 4) and GFP–annexin A5 (lane 5). The Western blotting used monoclonal antibody against annexin B1 or human annexin A5 followed by HRP-conjugated secondary antibody.


3.3 Translocation of GFP–annexin B1 after treatment with PMA

To examine the subcellular localization of GFP–annexin B1 in response to PMA, transfected SiHa cells were incubated in the presence or absence of 20nM PMA for 15, 30 or 45min. In cells that were transfected with an empty pEGFP vector, GFP were present unchanged after stimulation by PMA (Fig. 3A). Before stimulation, GFP–annexin B1 and GFP–annexin A5 were mainly present in nucleus, similar to that of the GFP control. However, after treatment with PMA, GFP–annexin B1 and GFP–annexin A5 showed a different pattern. After treatment for 45min, GFP–annexin B1 was translocated to the plasma membrane (Fig. 3B). In contrast, GFP–annexin A5 was mainly present on the granular structures throughout the plasma and homogeneously distributed on nuclear membrane (the fluorescence intensity ratio of nucleus vs. plasma was 9.32, 1.83, 1.74, and 0.96, respectively) (Fig. 3C).


Fig. 3

Subcellular localization of GFP, GFP–annexin B1 and GFP–annexinA5 in SiHa cells after treatment with PMA in the presence of calcium. SiHa cells were transfected with expression constructs encoding GFP (A) or GFP–annexin B1 (B), or GFP–annexin A5 (C) and processed for confocal laser scanning microscopy as described. The cells were fixed with 3.7% formaldehyde in the absence of PMA (1), after 15min treatment with PMA (2), after 30min treatment with PMA (3) and after 45min treatment with PMA (4). Before stimulation, GFP–annexin B1 and GFP–annexin A5 were mainly present in nucleus. After treatment for 45min, GFP–annexin B1 was translocated to the plasma membrane (B); By contrast, GFP–annexin A5 mainly present on the granular structures throughout the plasma and homogeneously distributed on the nuclear membrane (C). GFP proteins were dispersed unchanged before or after treatment with PMA.


3.4 Translocation of GFP–annexin B1 after stimulation by ionomycin

To investigate the subcellular localization of GFP–annexin B1 after increasing the intracellular calcium level, transfected SiHa cells were stimulated with 10μM calcium ionophore ionomycin in the presence of 2mM extracellular calcium. In the unstimulated cells, GFP–annexins B1 and GFP–annexin A5 were homogeneously located throughout the cells. In contrast to the treatment with PMA, these two proteins showed a similar distribution after ionomycin stimulation. After treatment for 30s, these two proteins were both translocated to the plasma and nuclear membranes (Fig. 4B,C). As a control, in cells transfected with empty pEGFP vector, the location of GFP was unchanged after treatment (Fig. 4A).


Fig. 4

Subcellular localization of GFP, GFP–annexin B1 and GFP–annexinA5 in SiHa cells after stimulation by ionomycin in the presence of calcium. SiHa cells were transfected with expression constructs encoding GFP (A), GFP–annexin B1 (B) or GFP–annexin A5 (C), and processed for confocal laser scanning microscopy. SiHa cells were fixed with 3.7% formaldehyde in the absence of ionomycin (1), after 10s (2) and after 30s stimulation by ionomycin (3). GFP–annexin B1 and GFP–annexin A5 were homogeneously located throughout the nucleus before treatment. After treatment, these two proteins were translocated to the plasma and nuclear membranes. As a control, GFP proteins were dispersed unchanged before or after treatment with ionomycin.


3.5 Changes in cytosolic Ca2+ concentration induced by PMA or ionomycin

Calcium-imaging studies were performed to determine the extent to which the calcium levels rose in response to be treated with PMA or ionomycin. The results showed the relative changes of intracellular fluorescence. For the pEGFP–annexin B1-transfected cells, the fluorescence intensity increased sharply to the maximal value (200±55% of the baseline, n=6) within 30s after treatment with ionomycin, the level remained constant (Fig. 5A, upper curve). However, it increased slowly and reached the maximal value (130±40% vs. the baseline, n=6) until 600s after stimulation by PMA (Fig. 5A, lower curve). In parallel studies, the fluorescence observed in pEGFP–annexin A5-transfected cells showed a similar pattern induced by PMA or ionomycin (Fig. 5B).


Fig. 5

Intracellular free calcium transients induced by ionomycin and PMA in SiHa cells transfected with pEGFP–annexin B1 and pEGFP–annexin A5. The cells were grown on glass-bottom dishes and loaded with rhod-2/AM. Changes in calcium levels were induced by perfusing the cell layer with ionomycin (10μM) or 20nM PMA in the presence of 2mM CaCl2 and were monitored using the confocal laser microscope. (A) Effect of pEGFP–annexin B1 transfected SiHa cells with ionomycin (upper curve) and PMA (lower curve) on the Ca2+ transient. (B) Effect of pEGFP–annexin A5 transfected SiHa cells with ionomycin (upper curve) and PMA (lower curve) on the Ca2+ transient.


4 Discussion

We have detected strong brown signals in the host-derived layer with granulomatous infiltration by using immunohistochemical staining. In addition, annexin B1was present both in cystic fluid of T. solium cysticerci and sera of infected humans/pigs. These findings indicate that annexin B1 can be detected outside the parasite, and may belong to the few members of annexins which can be secreted, as has been reported previously in studies of other members of this protein family (Christmas et al., 1991; Yeatman et al., 1993; Solito et al., 1994; Coméra and Russo-Marie, 1995; Thorin et al., 1995; Vergnolle et al., 1995; Siever and Erickson, 1997; Perretti, 1998).

Our previous study demonstrated an anti-PLA2 activity of annexin B1 by in vitro assay (Huang et al., 2005). Phospholipase A2 (PLA2) was the rate-limiting-step in the synthesis of lipid inflammatory mediators; the control of PLA2 activity was central to regulating the inflammatory process. In the present study, immunohistochemical analysis revealed that annexin B1 distributed in the host-derived bladder wall and especially in regions with high-grade inflammation, implying that annexin B1 might protect the parasite from the damage of the inflammatory response by inhibiting PLA2 activity.

In a previous study, we also found that apoptotic signals were detected from the metacestodes isolated from pigs that were inoculated with pcDNA3–annexin B1, but not in those injected with pcDNA3 (Wang et al., 2003). In cardiomyocytes, annexin A5 was externalized at an early stage of apoptosis and might have had a proapoptotic effect (Monceaua et al., 2004). In human neutrophils, a novel calcium-dependent proapoptotic effect of annexin A1 has been confirmed (Solito et al., 2003). It can be assumed that that annexin B1 might be secreted from T. solium cysticerci and then engulfed into the bladder wall cells to protect the parasite by its proapoptotic effect.

Further studies are needed to confirm these assumptions and to explore the real physiological roles of annexin B1. Annexins have been shown to bind to negatively charged phospholipids, such as those enriched in the inner leaflet of the plasma membrane and the cytoplasmic aspect cellular organelles such as secretary vesicles, in a calcium-dependent manner (Raynal and Pollard, 1994). In the present study, we generated chimeric constructs containing GFP tag, and monitored the intracellular localization of GFP–annexin B1 in living cells. Our previous study (Yan et al., 2002) has shown that annexin A5 is more homologous to annexin B1 than other human annexins, and therefore we selected it for parallel studies. The results show that GFP–annexin A5 are mainly located in the nucleus in unstimulated cells; after raising intracellular calcium by ionomycin, GFP–annexin A5 relocated from its homogeneous distribution in nucleus to the periphery of the nuclear and plasma membrane. These findings are consistent with those of previous studies (Sun et al., 1992; Koster et al., 1993), indicating that the GFP tag on the N-terminus had little influence on the function of annexins.

Our results indicated that GFP–annexin B1 and GFP–annexin A5 were both translocated to the plasma and nuclear membrane when intracellular Ca2+ was raised by ionomycin. However, GFP–annexin B1 and GFP–annexin A5 showed a different response to the stimulation of PMA. PMA stimulation not only elevated the intracellular Ca2+ but also increased PKC activity. PKCs are serine–threonine kinases that are activated by diverse stimuli, including mitogens, and participate in a variety of cellular processes (such as proliferation, differentiation and apoptosis) (Battaini, 2001; Nishizuka, 2001). Annexin B1 has a short motif “Thr-Ile-Thr” in its N-terminus, which is similar to “Thr-Val-Thr” in the N-terminus of annexin A5. Previous studies have shown that human annexins A1, A2, A3 and A4, Drosophila annexin B10 and Hydra annexin B12 possess a “Thr/Ser-Val/Ile-Lys/Arg” motif in their N-termini. This conserved motif has been proposed to be a PKC phosphorylation site (Raynal and Pollard, 1994; Rothhut, 1997). In contrast, in the N-termini of annexin A5 and A6, the third basic residue in the short motif has been substituted by an uncharged Thr residue; as a consequence, annexins A5 and A6 inhibit the protein kinase C activity via a mechanism of phospholipid sequestration (Dubois et al., 1998). Thus, we assumed that intracellular PKC activity increased upon PMA stimulation, and annexin B1 might inhibit the PKC activity by binding plasma membrane. However, the mechanism needs further investigation.

In conclusion, by using immunochemical analysis and ELISA assay, we first identified that annexin B1 could indeed be secreted from the cell. When the intracellular Ca2+ concentrations were raised by treatment with PMA or ionomycin, we found that GFP–annexin B1 was able to translocate from the cytoplasm to the plasma membrane or nuclear membrane. These findings shed light on further exploration of the secretion mechanism and real physiological roles of annexin B1.

Acknowledgments

We wish to acknowledge the financial support the grants from the National Natural Science Foundation of China (No. 30271167), and Key Fundamental Research Programs of Shanghai (No. 05JC14043) and National key fundamental research programs of “973” project (No. 2006CB504105).

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Received 24 March 2007/28 June 2007; accepted 27 August 2007

doi:10.1016/j.cellbi.2007.08.022


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)