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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Department of Molecular and Cell Biology, University of California, Berkeley, California
Submitted 19 March 2008 ; accepted in final form 8 May 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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stomach; kidney; intestine; membrane-cytoskeleton; phosphorylation
The intramolecular interaction of the N-term with C-term provides a means for regulating ezrin activity. In gastric parietal cells, where ezrin was first reported as a stimulation-dependent 80 K phosphoprotein (39, 40), N-C binding of ezrin was visualized by in vitro fluorescence resonance energy transfer (FRET) analysis in situ (48), complementing evidence from blot overlay experiments (14). Ezrin in the N-C conformation did not show significant binding to other proteins, because coimmunoprecipitation with anti-ezrin did not pull down any binding proteins from gastric gland lysates (48). Phosphorylation of ezrin on T567 was found to break the N-C binding and turn ezrin into the active conformation, which allows F-actin binding and stronger membrane binding (49). Similar observations were also observed with other members of the ERM protein family, moesin (20, 30, 34) and radixin (21, 25).
The development of a specific antibody against threonine-567-phosphorylated ezrin isoform (T567 in ezrin, T558 in moesin, and T564 in radixin) (25), together with the introduction of the T567D mutant (15), greatly facilitated the study of the physiological significance of this phosphorylation event. A common observation is that the phosphorylation on T567 is often accompanied by enhanced cellular activity, for instance, the translocation and activation of NHE3 in an intestinal cell line (36), and the formation of microvilli, lamellipodia, and membrane ruffles in several other systems (11, 15, 33). Naturally, phosphorylation on T567 is regarded as an activation mechanism for ezrin (12), and the phosphorylated ezrin is considered as an active form of ezrin (45).
The expression of ezrin T567D mutant also caused elongated projections on the plasma membrane of cultured gastric parietal cells (47). However, the elongated projections and the associated T567D mutant were not located on the apical membrane where the majority of native ezrin is usually located. Instead, T567D mutant expression caused abnormal growth of membrane projections on the basolateral membrane and thus changed the polarity of gastric parietal cells (47). We attributed this phenomenon to the blockage of dephosphorylation after our more recent discovery that there is a high turnover of phosphorylation on ezrin T567 (49). Fluorescence recovery after photobleaching (FRAP) analysis revealed that the ezrin T567D mutant was tightly associated with its basolateral membrane/cytoskeleton locus. Thus dephosphorylation is as important as the phosphorylation event for ezrin function in gastric parietal cells.
Is this phosphorylation turnover mechanism for the regulation of ezrin activity also present in other cell systems? We describe here surprising results, comparing the state of ezrin phosphorylation in gastric parietal cells and other freshly isolated epithelial cells, mainly renal proximal tubule cells and intestinal enterocytes, where ezrin is enriched in the brush border microvillar membranes. We found that the steady-state phosphorylation level of ezrin T567 in the latter brush border systems is much higher than that in gastric parietal cells; nevertheless, ezrin T567 phosphorylation is regulated by a turnover mechanism in all these epithelia. In addition, we provide evidence to support the notion that ezrin can bring membrane to filamentous actin binding sites, thus high turnover of ezrin phosphorylation in the actin binding domain empowers ezrin to reposition the membrane along the filamentous actin length and provide dynamic surface plasticity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The monoclonal anti-ezrin (4A5) antibody used in this study was purchased from Covance (Berkeley, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and HRP-conjugated goat anti-rabbit were purchased from Jackson ImmunoResearch (West Grove, PA). FITC-labeled phalloidin is from Sigma. Alexa 555-conjugated goat anti-mouse was a product of Invitrogen (Eugene, OR). Phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) rabbit antibody (anti-T567P) was purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti-moesin antibody is from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-actin (clone C4) was purchased from MP Biomedicals (Solon, OH).
Recombinant adenoviruses rAD/EzWT-CFP, rAD/EzT567A-CFP, and rAD/EzT567D-CFP were produced and amplified in our lab as described previously (47). These viruses express wild-type ezrin, its T567A mutant, or its T567D mutant, with a cyan fluorescence protein (CFP) tag inserted at the carboxy terminus.
Isolation of gastric glands, renal proximal tubules, renal proximal tubule cells, and small intestine organoids. All procedures and treatments for handling animals were reviewed and approved by the Berkeley Animal Care and Use Committee. Gastric glands were isolated from New Zealand White rabbits (Oryctolagus cuniculus) as previously described (47). About 60% of the mass of isolated gastric glands consists of parietal cells, and these contain virtually all of the glandular ezrin.
Isolation of the renal proximal tubules followed the procedure of Mandel's laboratory with modifications (9). Briefly, a kidney was taken immediately after the stomach was removed from the rabbit. After removal of the renal capsule, the cortex was trimmed from the excised kidney, minced, and digested for 30 min at 37°C in Eagle's minimum essential medium (MEM) supplemented with 20 mM HEPES (pH 7.3), 1 mg/ml collagenase, and 0.8 mg/ml bovine serum albumin (BSA). All solutions used for mincing, collagenase digestion, and subsequent centrifugations were all gassed with 100% O2 to minimize hypoxia during tissue preparation. The digested mixture was filtered through cheesecloth to remove large connective tissue and undigested materials and sedimented by centrifugation at 50 g for 2 min at room temperature. The brief centrifugation efficiently removed single cells, small fragments of tubules, and other small structures. The pellet was washed twice with oxygenated MEM (repeat resuspension and centrifugation) at room temperature. The pellet was then resuspended in oxygenated MEM supplemented with 20 mM HEPES and 4 mM glycine until ready for use. Microscopic examination of the preparation indicated that 
80% of the preparation is derived from proximal tubule. The remaining 20% consisted of other parts of the nephron and connective tissue, which have much lower levels of ezrin expression (3).
To isolate single proximal tubule cells, the isolated proximal tubules were first subjected to the same collagenase-enriched MEM described above. The digested materials were then filtered through 40 µM nylon cell strainers (Becton Dickinson, Franklin Lakes, NJ) to remove large tubules. The cells in the filtrate were pelleted by centrifugation at 200 g for 5 min at room temperature. Cells were washed twice with MEM before plating onto Matrigel (Collaborative Biomedical, Stony Brook, NY)-coated coverslips or dishes and incubated at 37°C in chemically defined culture medium A, which consists of DMEM/F12 (GIBCO BRL), 20 mM HEPES, 0.2% BSA, 10 mM glucose, 8 nM epidermal growth factor, 1x SITE medium (containing selenite, insulin, transferrin, and ethanolamine; S4920, Sigma), 1 mM glutamine, 100 U/ml penicillin/streptomycin, and 400 g/ml gentamycin sulfate, pH 7.4. After 5 h of incubation, the cells became attached to the vessel surfaces and adenoviral infection was started.
Approximately 30 cm of small intestine was removed from the rabbit, cut open, washed with saline, and the mucosa scraped. Fragments of mucosal scrapings were subjected to the same collagenase digestion procedure described for isolation of renal proximal tubules. The final pellet was resuspended in oxygenated MEM supplemented with 20 mM HEPES and consisted of small groups of villi that we call intestinal organoids.
Immunoblot analysis. Protein samples were separated by SDS-PAGE before transferring onto nitrocellulose membranes. Membranes were blocked with 2% BSA in Tris-buffered saline (10 mM Tris, pH 7.0, 150 mM NaCl) containing 0.05% Tween 20. The membranes were then probed with primary and secondary (HRP-conjugated) antibodies. Results were then recorded by X-ray films with the Western Lightning Chemiluminescence substrate (PerkinElmer Life Sciences, Boston, MA). When reprobing was needed, the blot was stripped (in 2% SDS, 1% 2-mercaptoethanol, and 62.5 mM Tris·HCl, pH 6.8) at 50°C for 30 min. The blot was then blocked and reprobed with another primary antibody. The signals from anti-T567P and anti-ezrin probing were found not to interfere with each other or subsequent Western blot results.
Ezrin extraction analyses with Triton X-100 and digitonin. Triton X-100 (TX-100) extraction analyses were performed to evaluate the association of ezrin with F-actin (9, 12). Freshly isolated gastric glands or renal proximal tubules were treated with 1 µM CLA for 5 min to block protein dephosphorylation and thus maximize phosphorylation levels, or treated with 10 mM sodium azide for 30 min to block metabolism and reduce ATP (and phosphorylation) levels. The tissues were then extracted with 1% TX-100 for 5 min at room temperature. After centrifugation at 100,000 g for 15 min at 4°C, samples of supernatant extracts and cytoskeleton pellet were collected and examined by Western blots successively probed to determine the relative phosphorylation level on ezrin T567 (T567-P), total ezrin, and actin.
Digitonin was used to perforate cells by way of cholesterol extraction for the study of cytosolic proteins with minimum interference to membrane proteins (24, 35). Freshly isolated gastric glands were compared with an experimental set treated with 1 µM CLA; control proximal tubules were compared with an experimental set treated with 10 mM NaN3. The respective glands and tubules were permeabilized by treatment with 40 µg/ml digitonin and extracted for 5 min, either in low-salt buffer (250 mM sucrose, 1 mM EDTA, and 10 mM HEPES, pH 7.3) or normal phosphate-buffered saline with 1 mM EDTA. Samples were centrifuged at 10,000 g for 5 min at 4°C. Blots of supernatant extracts and residual pellet fractions were probed for ezrin T567-P and ezrin. Additional gels were run in parallel for Coomassie blue staining.
Time course of the phosphorylation turnover on ezrin T567. Gastric glands aliquoted into individual tubes were treated with 1 µM CLA for various time periods as indicated. Aliquoted renal proximal tubules were treated with a membrane-permeable broad-spectrum kinase inhibitor, staurosporin, at 1 µM for various time periods as indicated. Reactions were stopped by immediate boiling in SDS-loading buffer. Samples were analyzed by Western blots probed for ezrin T567-P and ezrin.
Immunofluorescence microscopy. Proximal tubules and small intestine organoids treated with sodium azide and control samples were attached to the poly-L-lysine (P1399, Sigma)-coated coverslips, fixed by 3.7% formaldehyde, and permeabilized with 0.5% TX-100. Samples were then probed with anti-ezrin antibody. Afterward, the cells were incubated with Alexa 555-conjugated anti-mouse antibody together with FITC-phalloidin. Images of Alexa 555 (excitation with 543-nm laser, emission from 590–655 nm) and FITC (excitation with 488-nm laser, emission from 505–580 nm) were collected at 1 Airy unit pinhole with Plan-Neofluar x40/1.3 oil differential interference contrast objective on a Zeiss LSM 510 meta confocal microscope. Cells grown on Matrigel-coated coverslips were fixed by 3.7% formaldehyde and permeabilized with 0.1% TX-100, and the same procedure was then followed as described above.
Live cell imaging. Proximal tubule cells were grown on Matrigel-coated coverslips, infected with recombinant adenovirus expressing CFP-tagged wild-type ezrin, T567A mutant, or T567D mutant for 2 days. CFP images (excitation with 458-nm laser, emission from 473–515 nm) were then collected with a water immersion objective Achroplan x40/0.8 W at 1 Airy unit pinhole on a Zeiss LSM 510 meta confocal microscope.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Western blots of treated and control tissues were probed with anti-T567-P followed by stripping and reprobing with anti-ezrin (Fig. 1). Two proteins were revealed by T567-P antibody. The band with higher apparent molecular mass (Mr) is T567-P ezrin because it was recognized by a highly specific ezrin antibody. The other band is likely moesin judging from its slightly lower Mr and subsequent tests. Interestingly, the results for gastric glands differed markedly from those obtained for proximal tubules and small intestine. For gastric glands, the ezrin T567-P level increased greatly when incubated with CLA, indicating that T567 phosphorylation level is low in the native condition, whereas azide did not produce any detectable effect on ezrin T567-P level, again reflecting the fact that gastric ezrin carries a low level of T567-P in the steady state. A contrasting result was observed with renal proximal tubules. CLA had virtually no effect on ezrin T567-P level, whereas azide greatly depressed T567-P. The results for small intestinal enterocytes were similar to those for proximal tubules, indicating that both of these brush border-rich tissues have a higher steady-state level of ezrin T567-P than that of gastric glands.
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4 min; data not shown). Because of inherent problems with respect to permeation and multiplicity of action for the various inhibitors, these data cannot be taken as definitive values for reaction rates, but they do indicate that the T567-P turnover is relatively fast in these tissues.
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Ezrin-cytoskeleton binding is enhanced by phosphorylation. To evaluate T567-P turnover as a regulatory mechanism for ezrin activity in tissues, two extraction methods were used to study the interactions of ezrin with F-actin and plasma membrane in renal proximal tubules and gastric glands. Extraction with TX-100 removes soluble and membrane bound proteins, leaving behind the F-actin cytoskeleton pellet and many actin bound proteins (15). Isolated gastric glands and proximal tubules were taken fresh as control tissue, or treated with CLA to block protein dephosphorylation and thus maximize phosphorylation levels, or treated with sodium azide to reduce ATP phosphorylation levels. The tissues were then extracted with 1% TX-100 for 5 min at room temperature. Samples of supernatant extracts and cytoskeleton pellet were examined by Western blots probed for T567-P, total ezrin, and actin.
For gastric glands, total actin was predominantly distributed to the cytoskeleton pellet over the supernatant (4:1) and was not significantly different between the various experimental treatments (Fig. 3C). This is consistent with previous findings that actin in gastric glands is predominantly in the filamentous form (2). As expected, treatment with CLA resulted in a large increase (4–5 fold) in T567-P level compared with control glands (Fig. 3B). Treatment with N3 produced no significant change in T567-P compared with fresh glands (Fig. 3B). For all treatments, ezrin T567-P was always predominantly distributed toward the pellet, which was always about four times greater than that extracted into the TX-100 supernatant (Fig. 3B). In control glands, total ezrin was distributed between supernatant and pellet in approximately 60:40 ratio (Fig. 3A). Treatment with CLA greatly altered the TX-100 extraction so that the majority of ezrin (>80%) remained with the pellet (Fig. 3A). For glands treated with N3, there was very little difference from control, either with respect to the relative amount of T567-P or in the supernatant/pellet distribution ratio of total ezrin (Fig. 3A).
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For both gastric glands and renal proximal tubules, T567-P ezrin was always more distributed toward the cytoskeletal pellet than the TX-100 supernatant, consistent with studies on various cell lines (15, 25) and supporting the notion that T567 phosphorylation exposes the actin binding site on ezrin. The distribution data also predictably show that when T567 phosphorylation is high (e.g., CLA treatment in gastric glands and steady-state control for proximal tubules), total ezrin is also associated with the cytoskeletal pellet, and these trends are reversed on lowering phosphorylation with N3.
Ezrin remains bound to membrane in the nonphosphorylated form: biochemical evidence. In another extraction method, we used digitonin to form pores in the plasma membrane, allowing soluble cytoplasmic proteins to exit while retaining cytoskeleton, membrane proteins, and large protein complexes associated with membranes or cytoskeletal structures (24). Freshly isolated gastric glands were compared with an experimental set treated with CLA; control proximal tubules were compared with an experimental set treated with NaN3. The respective glands and tubules were permeabilized by treatment with digitonin and extracted for 5 min, either in low-salt buffer or normal phosphate-buffered saline. Blots of supernatant extracts and residual pellet fractions were probed for ezrin T567-P, total ezrin, and actin. Additional gels were run in parallel for Coomassie blue staining to ascertain that cytosolic proteins did leak out after digitonin permeabilization (data not shown).
Similar to earlier experiments, control levels of ezrin T567-P were relatively low in gastric glands and relatively high in proximal tubules; treatment of glands with CLA caused a large increase (4-fold) in T567-P (Fig. 4B), and treatment of tubules with N3 caused a large decrease in T567-P (Fig. 4D). When control preparations of either gastric glands or proximal tubules were permeabilized in low-salt buffer, relatively little of the total ezrin was released to the supernatant, e.g., <20% for glands and <5% for tubules (Fig. 4, A and C). There was little change in the pattern of ezrin released from permeabilized gastric glands after CLA treatment or the pattern of ezrin released by tubules after treatment with N3 (Fig. 4, A and C). These data contrasted sharply with those for TX-100 cytoskeletal extraction, where low activity of T567 phosphorylation was correlated with a higher distribution of total ezrin to the supernatant. Since control glands or N3-treated tubules demonstrate relatively little ezrin binding to actin cytoskeleton when the bulk of ezrin is in the dephosphorylated state, the general retention of ezrin in the digitonin-permeabilized preparations appears to be due to another binding site, likely the NH2-terminal membrane binding site.
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Ezrin remains bound to membrane in the nonphosphorylated form: imaging evidence. To study the localization of nonphosphorylated ezrin in an in situ setting, renal proximal tubules were incubated either with nitrogen for hypoxia or with sodium azide for chemical anoxia before immunofluorescence staining with ezrin antibody. Phosphorylation on ezrin T567 was found greatly decreased by hypoxia treatment and further decreased by anoxia (NaN3) treatment (Fig. 5A). Nevertheless, proximal tubules in each of the three different treatments were stained similarly (Fig. 5, B–D). There was always a high degree of colocalization of ezrin with F-actin. Most of the staining was with the apical brush border membrane facing the lumen of the tubule; a weak but clear staining of the basal membrane was also detected; cytoplasm and lateral membrane were not stained. These results indicated that ezrin is localized to a membrane surface regardless of the phosphorylation level on T567.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Ezrin from both renal proximal tubule cells and intestinal enterocytes were found to be highly phosphorylated, compared with the low steady-state level of phosphorylation in the nonsecreting gastric parietal cells (Fig. 1). Both the renal proximal tubule cells and small intestinal enterocytes are known for the presence of their characteristic apical brush border membranes, which are densely packed with uniformly sized microvilli. In contrast, the microvilli found in nonsecreting gastric parietal cells normally exist in various lengths and sparse distribution (13). Upon stimulation, there is a massive membrane recruitment from intracellular tubulovesicles onto the apical canalicular membrane, forming a much denser distribution of elongated apical microvilli (13). Previously, we reported a significant increase of ezrin phosphorylation (40) and, specifically, phosphorylation of ezrin on T567 (49), in gastric parietal cells when these cells were physiologically stimulated. Thus, a higher steady-state level of ezrin T567-P seems to be correlated with a denser distribution of microvilli, whether from the same cell (e.g., the different physiological conditions of parietal cell) or from different types of tissue.
T567 phosphorylation turnover is required for ezrin function in renal proximal tubule cells. The prompt decrease in ezrin T567-P with azide treatment in renal proximal tubule cells and small intestine enterocytes indicates that continuous kinase activity is needed to maintain the high level of phosphorylation on ezrin T567. The importance of T567-P turnover for the regulation of ezrin function in renal proximal tubule cells is demonstrated with the expression of the T567D mutant, which mimics permanent phosphorylation of T567, not allowing the turnover of ezrin between phosphorylated and dephosphorylated forms. When T567D mutant ezrin was overexpressed in primary kidney cell cultures, surface membrane structures were abnormally long and tended to accumulate at a small area on the plasma membrane, where the T567D mutant was localized. This morphological change obviously affected the fine structure of microvilli and may even disrupt the polarized distribution of the membrane proteins. Since the precise localization of channels and pumps on the plasma membrane(s) is essential for the functional transport of nutrients from the glomerular filtrate back into circulation, it is conceivable that the abnormal morphology of the renal proximal tubule cells would result in a decreased efficiency of function. Unfortunately, this experiment cannot be done with the primary cultures of proximal tubule cells expressing T567D mutant since the cells do not maintain their polarity in culture. However, experiments done with a polarized kidney epithelial cell line NRK-52E demonstrated an abnormal relocation of Na-K-ATPase onto the apical membrane upon treatment with RhoA (23), which is known to induce phosphorylation of ERM proteins on the conserved T567/T564/T558 site (25, 33).
The T567-P high turnover mechanism for regulation of ezrin activity is thus not limited to gastric parietal cells but also applies to ezrin in renal proximal tubule cells and small intestine enterocytes. In addition, moesin, which is expressed at relatively high levels in renal proximal tubules, was also subjected to the high turnover regulation on the phosphorylation of the conserved T558 (Fig. 2B). Thus it may be that this high turnover mechanism is a universal one for the regulation of all ERM protein activity.
Ezrin brings membrane to cytoskeleton. Studies with transformed cell lines often described ezrin as a diffusely localized, or cytosolic, protein, in its inactive form (4, 5, 42). Since ezrin has increased membrane and cytoskeleton binding affinity upon activation (by phosphorylation), the major function of ezrin was believed to be "linking F-actin to membrane" (6). However, our examination of the state and localization of ezrin in three freshly isolated tissues suggests a modification of this concept. Phosphorylation on T567 was observed to induce tighter membrane binding of ezrin in gastric parietal cells (49), as expected from many other studies (12, 19, 45), since the membrane-binding NH2 terminus is partially masked by the COOH terminus in the nonphosphorylated N-C binding conformation (14, 48). However, ezrin in its nonphosphorylated form still localizes to the microvilli-rich apical membrane of parietal cells as shown previously (49) and in Fig. 4A in the present study. Similarly, in renal proximal tubule cells and small intestine enterocytes, the dephosphorylated form of ezrin remains localized on the brush border microvilli-rich membrane (Figs. 5 and 6). Binding analyses showed that dephosphorylation by anoxia treatment would only slightly shift the distribution of ezrin toward the cytosolic pool, which is very small (Fig. 4C). Severe alterations in the cytoskeleton and the repolarization of many membrane proteins occur rapidly after ischemia and reperfusion in the kidney (26–29). Although the mechanism of these transitions is not completely understood, it is known that ischemia alone does not cause the rearrangement of F-actin cytoskeleton. While reperfusion after 40 min of ischemic treatment caused significant decrease in F-actin, the remaining F-actin signal still largely remained on the brush border membrane (7). Our procedures to induce anoxia or hypoxia, and the observed results, were obviously similar to the renal ischemia treatments without reperfusion.
The binding of T567 unphosphorylated ezrin to the membrane seems to be mediated by PIP2. Tsukita and colleagues (44) found that treatment of two cell lines A431 and MDCK II with staurosporin could decrease the T567-P without affecting the membrane localization of ezrin. However, when the PIP2 levels in A431 cells were decreased by microinjection of C3 transferase (which blocks the activation of Rho and PI4,5-kinase), ezrin was found dephosphorylated on T567 and diffused away from microvillus membranes. The PIP2 level in MDCK II did not respond to C3 transferase but could be blocked with neomycin, which also caused a decreased T567-P level and diffusive staining of ezrin. The dependence of ezrin function on PIP2 binding was also reported by Arpin's group (12). Their results with the LLC-PK1 cell line indicated that ezrin binding to PIP2, through its NH2-terminal domain, is required for membrane localization and T567 phosphorylation. It may well be that the membrane binding site on ezrin has some dependence on ionic bonding because the depletion of ezrin in permeabilized glands and tubules was increased by high salt (Fig. 3), consistent with previous observations in gastric glands (18), and as might be inferred by cytosolic divalent ion competition for ezrin release in glands (43) and in LLC-PK1 cells (12); however, in the latter case the authors have suggested that increased cytosolic Ca2+ might catalyze PIP2 hydrolysis.
In contrast to the continuous binding to the membrane, the binding of ezrin to F-actin was greatly enhanced when the COOH-terminal actin-binding site was unmasked by phosphorylation at T567. Mandel et al. (9) demonstrated that dissociation of ezrin from actin cytoskeleton occurred in renal proximal tubules upon anoxia treatment. They later showed evidence that anoxia treatment induced ezrin dephosphorylation by two-dimensional electrophoresis (8). Very likely this dephosphorylation occurs on T567, although we cannot exclude the possibility of other Ser/Thr phosphorylation site(s). By F-actin cosedimentation assays (49) and Triton extraction analyses (Fig. 3), dissociation of ezrin from F-actin was found to be induced by dephosphorylation on T567 in gastric parietal cells. Similar data were obtained from many other cell models (12, 20, 30, 31, 37, 45, 49).
A relatively constant membrane binding and the ever changing F-actin binding gives the ezrin molecule the power to associate and reassociate membrane and cytoskeleton, thus enabling a dynamic reposition of membrane along the filamentous actin length, in a fashion similar to a rolling molecular motor attached to cell membrane. A cartoon depicting the relaxation-reattachment hypothesis to promote dynamic rearrangement within defined surface morphology is shown in Fig. 9. Such a model is logical and essential for surface plasticity in view of the growth and repositioning required for cytoskeleton expansion, as well as the reestablishment of membrane surface forces as processes of recruitment and endocytosis occur. This model also explains why blocking moesin dephosphorylation with CLA caused the impairment of neutrophil motility (46), which is crucial to effective host defenses against microorganisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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