Journal of Undergraduate Research
Volume 7, Issue 4 - March/April 2006

Expression and Localization of Dynamin II in Osteoclasts

Kristie Johnson

ABSTRACT

Osteoclasts are hematopoetic cells that are specialized to resorb bone. The pathogenesis of a number of diseases including osteoporosis, rheumatoid arthritis and metastatic bone cancer involves misregulation of osteoclasts. Osteoclastic bone resorption requires reorganizations of the cytoskeleton and extensive membrane trafficking. Dynamin II has emerged as a key integrator of cytoskeletal and membrane dynamics and has been reported to be present in osteoclasts. In this study, we have confirmed and expanded on previous reports. We find that dynamin II is present in osteoclasts, but is not upregulated during osteoclastogenesis. We detected dynamin II in the actin rings of osteoclasts. Dynamin II was expressed at similar levels in osteoclasts compared with tumor cells that utilize dynamin II for metastasis. Although the function of dynamin I has been tied to lipid rafts, we found little dynamin II associated with rafts isolated from osteoclasts. Dynamin II was pulled down with vacuolar H+-ATPase, a major element of the membrane vesicles that are moved in osteoclasts in preparation for resorptive activity, in immunoprecipitations assays. These data are consistent with dynamin II playing a role in integrating membrane and cytoskeletal dynamics in osteoclasts.

INTRODUCTION

The guanosine triphosphatase (GTPase) dynamin II is involved in the formation of tubules and vesicles during endocytic trafficking and the fission of endocytic vesicles from the plasma membrane (McClure and Robinson, 1996). Dynamin II may also be involved with remodeling of the actin cytoskeleton (Schafer et al., 2002). Recent evidence in studies involving osteoclasts suggests a possible role for dynamin II in osteoclast function (Ochoa et al., 2000; Mulari et al., 2003). These reports differ regarding the localization of dynamin II. Ochoa and colleagues indicate that dynamin II is associated with the podosomes that compose actin based structures called actin rings (Ochoa et al., 2000). By contrast, Mulari and colleagues find dynamin II associated with the ruffled plasma membrane, but not podosomes (Mulari et al., 2003). Podosomes are unique structures that are composed of microfilaments and are found in cell types that are involved in the invasion of cells through matrix. Metastatic tumor cells, for example, form podosomes that are involved in providing them with the capacity to invade tissue.

Osteoclasts are the cells specialized for mineralized bone resorption (Teitelbaum, 2000). Osteoclasts are found in two forms, the inactive (non-resorbing) and the active (resorbing form). In order for inactive osteoclasts to become active, V-ATPases must be transferred from the cytosol into the ruffled plasma membrane, a domain of the plasma membrane that apposes the bone that is to be resorbed. Osteoclasts express podosomes and use them in bone resorption, which can be thought of as a specialized form of tissue invasion. Osteoclasts invade mineralized tissue and destroy both the extracellular matrix proteins and solubilize the mineral. To accomplish these functions, which are necessary for the health of the bone and for metabolic calcium regulation, podosomes are organized into structures called actin rings (King and Holtrop, 1975). Recent data suggest that podosomes in osteoclasts are very dynamic in that the actin is constantly polymerizing and depolymerizing (Saltel et al., 2004; Destaing et al., 2003).

Cortactin is a well-characterized biochemical link between dynamin and the actin filament assembly (Schafer et al., 2002). Cortactin interacts with the actin-related protein 2/3 (Arp2/3) complex and neural Wiscott-Aldrich Syndrome protein to regulate actin polymerization (Weaver et al., 2001). The Arp2/3 complex is required for actin ring formation in osteoclasts (Hurst et al., 2004). Cortactin is upregulated during osteoclastogenesis (Hiura et al., 1995). Recent data indicate that podosomes are involved in the targeting of Vacuolar H+-ATPase (V-ATPase), a membrane-associated enzyme vital for osteoclast function, during the activation of osteoclasts (Lee et al., 1999; Chen et al., 2004). In addition, recently the Holliday laboratory recently detected a potential interaction between V-ATPases and dynamin II in a proteomics assay (L. S. Holliday, personal communication, June 1818 2005). Taken together, these data suggest that dynamin II may play an important role in coupling cytoskeletal and membrane dynamics in osteoclasts.

In this study we examined the expression, localization and protein interactions of dynamin II in osteoclasts. Our data support the hypothesis that dynamin II may contribute to the integration of actin polymerization with membrane dynamics.

MATERIALS AND METHODS

Reagents

Unless otherwise noted materials were obtained from the Sigma Chemical CO (St Louis, MO) and were of the highest analytical grade. Anti-dynamin II antibody was from BD Transduction Laboratories (Cat # D27120-050). Fluorescent second antibodies were from Jackson Immunoresearch.

Generation of Osteoclasts

Mouse marrow osteoclasts were generated as described previously (Holliday et al., 1995). 8-20 gm Swiss-Webster mice were killed by cervical dislocation, femora and tibia were dissected from adherent tissue, and marrow was removed by cutting both bone ends, inserting a syringe with a 25 gauge needle and flushing the marrow using alphaMEM plus 10% fetal bovine serum (alphaMEM D10). The marrow was washed twice with alphaMEM D10 plus 10-8 M 1, 25-dihydroxyvitamin D3 (calcitriol). Cultures were fed on day 3, replacing half the media per plate and adding fresh calcitriol. After 5 days in culture, osteoclasts appeared. These were detected as giant cells that stained positive for tartrate-resistant acid phosphatase activity (TRAP; a marker for mouse osteoclasts) and overexpressed V-ATPase subunits. The University of Florida Institutional Animal Care and Usage Committee approved this protocol.

Osteoclast-like cells were differentiated from RAW 264.7 cells, a mouse hematopoietic cell line, by stimulation with recombinant RANK-L, as described(Hurst et al., 2004). In both cases, once mature, the osteoclasts or osteoclast-like cells were scraped and replaced on either glass coverslips or bone slices.

Western Blot

Proteins were separated by electrophoresis on 12% Acrylamide (40% stock, 29:1) gels, then transferred to nitrocellulose and blocked using blocking buffer (20 mM, Tris/HCl, 100 mM NaCl, 0.5% BSA, 0.05% Tween 20, pH 7.5) for 2H. Membranes were probed with primary dynamin II antibody at a dilution of 1:1000 for 2 hours, then washed in blocking buffer. Polyclonal anti-mouse horseradish peroxidase (1:1000) was used as secondary antibody treatment for 1hour and then blots were washed. The proteins were detected with Supersignal West Dura Extended Duration Substrate kit (Fisher, PIA-34075, or PIERCE, 0034075) following the manufacturer’s instructions and bands were visualized using an Alpha Innotech chemiluminescense detector.

Dot Blot

Lipid rafts were obtained from Raw 264.7 osteoclast-like cells, as described previously (Ha et al., 2003). Lipid raft fraction samples were taken and diluted to three degrees: 1x dilution, 1/10 dilution, and 1/100 dilution. Each of the 35 fractions was diluted accordingly, and 2μl was spotted onto nitrocellulose membrane. After all samples/dilutions were spotted and allowed to dry, the membranes were probed with antibodies as described above.

RESULTS

Osteoclasts differentiate in mouse marrow cultures in response to treatment with calcitriol. Western blots of cultures, plus or minus calcitriol, showed no-difference in levels of dynamin II. (Figure 1). We were interested in comparing the relative levels of dynmin II in osteoclasts with 4T1 cells, a mouse metastatic tumor cell line thought to utilize dynamin II in the metastatic process. Initial Western Blots of the Raw 264.7 osteoclasts and 4T1 tumor cells probed with Anti-dynamin II, suggested that there was a significantly higher expression of dynamin II in the tumor cells (Figure 2a). To further investigate this observation, a series of dilutions was performed on the tumor cell samples. The result of the blot showed that the Western blot technique was detecting dynamin II in a strikingly non-linear manner. The actual levels of dynamin II (2b) and actin (2c) were very similar in osteoclasts and 4T1 cells.

Figure 1. Expression of dynamin II during osteoclastogenesis

Figure 1: Expression of Dynamin II during osteoclastogenesis (+/- D3). Mouse marrow cultures were grown in the presence or absence of calcitriol (D3+/-) , protein was extracted, seprated by SDS-PAGE and blotted. Western Blots were probed with Anti-dynamin II (left) and duplicated gels were stained with Coomassie Brilliant Blue to detect total protein (right). Dynamin II, molecular weight 100, is present in osteoclasts but not upregulated in response to osteoclastogenesic stimuli

 

Figure 2. Comparison of dynamin II in OC vs. 4T1 tumor cells

Figure 2: Comparison Dynamin II in OC vs. 4T1 Tumor Cells. Figure 2a: Western Blot suggests higher levels of dynamin expression in 4T1 cells than in the RAW osteoclasts. Figure 2b: Dilution of 4T1 cells for comparison with RAW 264.7 cells. Figure 2c: Stardardization with by blotting with an anti-actin antibody. The actin to dynamin ratio is similar.

Confocal microscopy was used tovisualize the distribution of dynamin II in osteoclasts by immunocytochemistry. Dynamin II was associated with the actin rings of the RAW 264.7 osteoclasts, and was present in a diffuse distribution in the 4T1 cells (Figure 3).

Figure 3. Distribution of dynamin in osteoclasts by immunocytochemistry

Figure 3: Distribution of Dynamin in Osteoclasts by Immunocytochemistry. RAW 264.7 cells (left), show dynamin II expression in actin rings (arrows). 4T1 tumor cells (right), show diffuse dynamin expression. micrographs are to same scale.

To test whether dynamin II was concentrated in lipid rafts derived from osteoclasts, a dynamin II dot blot technique was developed. We found little dynamin II associated with rafts isolated from osteoclasts (Figure 4). By contrast, cortactin another cytoskeletal protein that interacts with dynamin II, was found at higher concentrations associated with the rafts.

Figure 4. Dynamin II and cortactin in lipid raft (dot blot)

Figure 4. Dynamin II and Cortactin in lipid raft (Dot Blot). Membranes from Raw 264.7 osteoclasts were isolated, extracted with 1% Triton X-100 and membranes were floated on a sucrose step gradient (5%/35%/80%) by centrifugation at 125,000 xg for 24 H. 0.5 ml sample were collected and samples werespotted onto membranes as described in Materials and Methods. Little dynamin II associated with rafts isolated from osteoclasts. Larger amounts of cortactin, a dynamin II binding protein, was recovered in the rafts fraction.

To confirm that dynamin II interacts with V-ATPases, immunoprecipitations of dynamin II from osteoclast extracts were performed using an anti-V-ATPase polyclonal antibody. Despite extensive washes of the precipitated beads, dynamin II was detected.in the vacuolar H+-ATPase immunopprecipitates (Figure 5).

Figure 5. Dynamin immunoprecipitation (IP)

Firgure 5: Dynamin Immuno-precipitation (IP). Raw 264.7 osteoclast were homogenized with 1% Triton X-100 and spun at 100,000 xg for 60 min . 20 microliters of polyclonal anti-V-ATPase antiser was added to 1 ml of extract. After 1H incubation, 50 microliters of sepherose-protein A beads were added and incubated on a rotator for 10 min. The beads were spun down at 10,000 xg for 5 minutes and washed 3X with PBS. Proteins were removed from the beads with 50 microliters SDS-PAGE sample buffer. Proteins were boiled, subjected to SDS-PAGE, blotted to nitrocellulose and probed with anti-dynamin II antibody. Chemiluminscent detection of Dynamin II (left) is shown on the IP lined up with the same blot detected by light (the molecular weight marker were pre-stained standards. Dynamin II was detected at the correct MW at of 100 kD.

DISCUSSION

These data are consistent with dynamin II paying a role in integrating membrane and cytoskeletal dynamics in osteoclasts. Dynamin II was detected in osteoclasts at levels similar to 4T1 tumors cells where dynamin II is thought to play an important role. Dynamin II was found in the specialized actin ring structure of osteoclasts. In addition, dynamin II was detected in immunoprecipitates of V-ATPase from osteoclasts. This result was confirmatory to other data obtained from another type of experiment (Vergara and Holliday, Unpub. Data).

Although dynamin II likely plays an important role in osteoclasts it was no upregulated during osteoclastogenesis. Certain vital proteins for osteoclast function, including V-ATPase subunits and cortactin, are dramatically upregulated during osteoclastogenesis. Upregulation of proteins that interact with dynamin II like cortactin and V-ATPases may may provide new binding partners that enable dynamin II to play a new specialized role in osteoclasts.

It was also somewhat surprising that dynamin II was enriched in the lipid raft fraction; the rafts may have been isolated from inactive osteoclasts. The type of raft with which dynamin II interacts had not yet formed. Isolation of rafts from actively resorbing osteoclasts will be required to test that idea.

In summary, we found dynamin II to be present in osteoclasts where it is concentrated in actin rings. We detected an association with V-ATPase. These data support the hypothesis that dynamin II may serve to integrate actin dynamics with vesicle transport in osteoclasts.

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