Article |
Address correspondence to James R. Bartles, Dept. of Cell and Molecular Biology, Ward Building 11-185, Feinberg School of Medicine, Northwestern University, 303 East Chicago Ave., Chicago, IL 60611. Tel.: (312) 503-1545. Fax: (312) 503-7912. email: j-bartles{at}northwestern.edu
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Abstract |
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Key Words: microvilli; stereocilia; hair cell; deafness; jerker
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Introduction |
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PABs are known to contain different complements of actin-bundling proteins (Bartles, 2000), but little is known about what each contributes. For example, enterocyte brush border microvilli contain villin, fimbrin, and espin (Bartles, 2000), whereas hair cell stereocilia contain fimbrins (Tilney et al., 1989; Daudet and Lebart, 2002) and espin (Zheng et al., 2000). Consistent with the idea that each actin-bundling protein plays an important and specific role, mutations that impair or eliminate different actin-bundling proteins have distinguishable effects on PAB organization (Tilney et al., 1998; Ferrary et al., 1999; Zheng et al., 2000).
There are many indications that the lengths of microvilli and stereocilia, and hence their PAB scaffolds, are tightly regulated. Microvilli and stereocilia grow to markedly different lengths, even though both originate as microvillus-like precursors that emanate from electron-dense patches beneath the plasma membrane (DeRosier and Tilney, 2000). Microvillus length varies in a regular way among cell types, ranging from the 12-µm long, uniform brush border microvilli typical of enterocytes (Mooseker, 1985) to the 80-µm long microvilli of epithelial cells lining the human caput epididymidis (Pacini et al., 1980). Stereocilia also exhibit characteristic differences in length as a function of hair cell position in the inner ear. For example, the length of the tallest stereocilia on outer hair cells in the hamster increases gradually from 1.2 µm at the base of the cochlea to 5 µm at the apex (Kaltenbach et al., 1994). In addition, precise variations in stereocilium length are present within the highly organized staircase array of stereocilia atop each hair cell (Tilney et al., 1992) and may be required for mechanosensory transduction (Pickles and Corey, 1992).
Increases in PAB length are realized during distinct phases of development and appear to be accomplished by two different mechanisms (DeRosier and Tilney, 2000). Invertebrates rely heavily on the end-to-end joining of relatively short bundles to make long PABs of a modular design (DeRosier and Tilney, 2000). However, a modular organization has never been reported for vertebrates. This suggests that their PABs become longer principally through the further elongation of the filaments in a bundle. Little is known about how this elongation might be accomplished and what molecules might be involved.
Recent studies of GFP-actin in transfected cells support the notion that the actin filaments of PABs in the brush border microvilli of LLC-PK1-CL4 (CL4) cells (Tyska and Mooseker, 2002) and in the stereocilia of cochlear hair cells (Schneider et al., 2002) exhibit actin treadmilling. This would imply that PAB length reflects a balance between ongoing actin polymerization and depolymerization reactions in a bundle and raises the intriguing possibility that PAB length needs to be maintained throughout the life of a PAB.
We have identified and characterized espin actin-bundling proteins in the PABs of Sertoli cell junctions (Bartles et al., 1996; Chen et al., 1999), brush border microvilli of enterocytes and renal proximal tubular epithelial cells (Bartles et al., 1998), and stereocilia of hair cells in the inner ear (Zheng et al., 2000). In addition, we have determined that the espin gene is the target of the jerker deafness mutation in mice (Zheng et al., 2000). A characteristic property of the espins is that they bind to and cross-link actin filaments with a 10100-fold higher affinity than other actin-bundling proteins (Kd = 70220 nM, depending on espin isoform; Chen et al., 1999). To determine whether espins affected PAB length and actin dynamics in PABs in vivo, we investigated the effects of espins in a well-defined model system, the brush border microvilli of CL4 cells.
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Results |
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Effect of espin on the length of CL4 cell microvilli and their PABs
After 10 d in culture, we transfected CL4 cells with a plasmid containing espin cDNA under the control of the cytomegalovirus (CMV) promoter. When transfected cells were examined 1724 h later, the GFP-espin fusion protein was highly concentrated in the microvilli, which showed dramatic increases in length (Figs. 1 and 2). The localization of GFP-espin in the microvilli was indistinguishable from that of F-actin as revealed by double labeling with fluorescent phalloidin (Fig. 1), suggesting that the increased length of the GFP-espinlabeled microvilli mirrored a comparable increase in the length of the PAB at their core. Both fluorescent probes were distributed continuously along the length of the microvilli. Microvillar PAB length measured from oriented confocal z-sections was 7.90 ± 0.09 µm (mean ± SEM; n = 555 microvilli, 28 cells) in the espin-expressing cells versus 1.33 ± 0.04 µm (n = 189 microvilli, 14 cells) in neighboring untransfected control cells (Fig. 2). The GFP tag did not influence the outcome, because microvillar length increased to the same extent when espin was expressed in untagged form and localized by immunofluorescence.
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The length of the microvillar PAB was correlated positively with espin protein expression level over the range examined (Fig. 2, A and B). Significant differences from control were observed even when espin was expressed at the 2% level (1.95 ± 0.03 µm; n = 197, 12 cells). Between the 10 and 100% levels, the response appeared roughly linear, a 10-fold increase in espin level resulting in an 2.5-fold increase in length.
Espin gradients among cochlear hair cells
We noted a similar positive correlation between espin level and stereocilium length among cochlear hair cells in situ. Cochlear hair cells exhibit a gradual approximately threefold increase in stereocilium length from base to apex along the cochlear spiral (Kaltenbach et al., 1994). We isolated cochlear whole mounts from adult rats and labeled them by immunoperoxidase or immunofluorescence using espin antibody after fixation and permeabilization with nonionic detergent. With either method, we consistently observed an increasing gradient of signal intensity from base to apex along the cochlear spiral. Fig. 4 shows representative confocal images, collected using identical settings, of the apical (A and B), middle (D) and basal (F) turns of a single cochlear whole mount as depicted in the diagram (top). The endogenous espin of hair cells is concentrated in the stereocilia; for each cochlear region, a single row of inner hair cells and three rows of outer hair cells can be seen. To the right of each confocal image is a color-scale plot of fluorescence intensity (Fig. 4, C, E, and G). This exposure highlights differences in intensity among the outer hair cells from the three regions. Using estimates of fluorescence intensity computed from pixel values, the outer hair cells showed an approximately threefold average increase in fluorescence intensity from base to middle and an additional approximately threefold average increase in intensity from middle to apex.
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Espin domains involved in the lengthening effect
To identify the parts of espin required for the microvillar PAB lengthening effect, we transfected CL4 cells with truncation or deletion constructs that lacked known structural or functional domains. A number of espin domains could be eliminated without reducing lengthening, such as the proline-rich peptides (Fig. 5, ce), the 28-aa additional F-actinbinding site (Fig. 5 f), or the WASP homology 2 (WH2) consensus domain (NSELLAEIKAGKSLKPT; Fig. 5 g). All of these mutated espin proteins were expressed at a similar level, became highly concentrated within microvilli, and increased average microvillar PAB length to 7.47.9 µm. The only mutations that made a major difference were those affecting the espin COOH-terminal peptide (Fig. 5, ik, asterisks). This peptide contains the actin-bundling module that is shared among known espin isoforms and is necessary and sufficient for potent actin-bundling activity in vitro (Bartles et al., 1998). Deletion of the actin-bundling module (C117; Fig. 5 i) reduced microvillar PAB length to near control levels, even though Western blots of replicate dishes of cells transfected with espin or the
C117 construct showed similar levels of protein of the expected molecular mass. The
C117 construct did not become concentrated in microvilli, but was distributed diffusely throughout the cytoplasm. Espins, which are monomeric in solution, are believed to derive actin-bundling activity from two F-actin-binding sites disposed roughly at opposite ends of the actin-bundling module (Bartles et al., 1998; Chen et al., 1999). Consistent with a requirement for these two F-actin-binding sites, deletion of either end of the actin-bundling module also reduced microvillar PAB length to near control levels (Fig. 5, j and k) and blocked targeting to microvilli without affecting protein expression levels (Fig. 5, inset at top).
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GFPT-fimbrin was expressed at an average level that was 80% of that of GFP-espin (Fig. 6 B, bottom left) and increased microvillar PAB length to 2.60 ± 0.03 µm (n = 254 microvilli, 15 cells). When compared with the lengthening effect expected for GFP-espin expressed at the 80% level (Fig. 2 B), the relative microvillar PAB lengthening activity of T-fimbrin was estimated to be 22% of that of espin (Fig. 6 C). CL4 cells transfected with GFPI-fimbrin plasmid showed no differences from controls, but we could not achieve high enough levels of protein expression to allow for adequate comparison.
GFP-villin was expressed at an average level that was 30% of that of GFP-espin (Fig. 6 B, bottom right) and increased microvillar length to 3.57 ± 0.06 µm (n = 197 microvilli, 13 cells). When compared with the lengthening effect expected for GFP-espin expressed at the 30% level (Fig. 2 B), the relative microvillar PAB lengthening activity of villin was estimated to be 70% of that of espin (Fig. 6 B). Similar results were obtained using GFP-tagged or untagged chicken villin and an untagged version of mouse villin. Compared with the long microvilli elicited by espin (Fig. 6 D), those formed in response to villin appeared more numerous and thin (Fig. 6 G).
Effect of low doses of cytochalasin D
Low doses (20200 nM) of cytochalasin D block actin polymerization by capping filament barbed ends (Sampath and Pollard, 1991), without causing the secondary effects, such as binding to monomer and nucleation of actin polymerization, that are observed at concentrations in the micromolar range (Goddette and Frieden, 1986). We compared the effects of 100 nM cytochalasin D on microvillar PAB length in transfected CL4 cells expressing small espin or espin at the 40% level. These two constructs resulted in similar levels of protein expression and increased microvillar PAB length three- to fourfold in the absence of cytochalasin D (Figs. 2 and 6). We added cytochalasin D immediately after transfection and measured microvillar PAB length 1213 h later. The 100 nM cytochalasin D inhibited the microvillar PAB lengthening response to small espin and espin by an average of 86 ± 2% and 80 ± 2%, respectively (n = 170213 microvilli, 1012 cells). This suggested that the espin-mediated lengthening response required additional actin polymerization predominantly, if not exclusively, at filament barbed ends. Beyond having shorter microvilli, the cells treated with 100 nM cytochalasin D showed no signs of morphological deterioration. The inhibitory effect was reversible; cells recovered long microvilli within 4.5 h after removal of the drug.
Effect of espin on actin depolymerization in vitro
We assumed initially that espin increased PAB length because its high-affinity cross-links caused a pronounced slowing of actin depolymerization. However, in two different in vitro assays, we found that the presence of bound 6X His-tagged espin at a level predicted to achieve maximum cross-linking (approximately one espin bound/four actin monomers; Chen et al., 1999) slowed actin depolymerization <2-fold.
In one assay, we examined the rate of release of actin monomer into the high-speed supernatant as a new steady-state was approached after the addition of 10 µM of the actin monomer-sequestering drug latrunculin A to preformed filaments. Densitometric analysis of the gels showed that the presence of espin cross-links caused only a slight (1.3-fold) decrease in the rate of monomer release (Fig. 7 A). At the end of the experiment,
50% of the actin monomer present initially in filaments had been released into the supernatant.
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Effect of espin on treadmilling in microvillar PABs: identification of actin monomer-binding and profilin-binding domains
Next, we used FRAP to determine whether espin affected treadmilling dynamics in the long microvillar PABs of espin-expressing CL4 cells. Cells were cotransfected with plasmids encoding untagged espin and GFPß-actin. The expression of the GFPß-actin had no effect on microvillar PAB length when expressed in the absence of espin. In initial experiments, we could find examples of a rapid recovery of GFPß-actin fluorescence at the tip of photobleached long microvilli. However, an unexpectedly rapid recovery of GFPß-actin fluorescence throughout the entire microvillus frequently obscured the site of initial recovery and consistently precluded efforts to monitor any subsequent tip to base movements of the zone of recovered GFPß-actin fluorescence (Fig. 8 A).
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CL4 cells were examined by FRAP either 4 h or 24 h after cotransfection with GFPß-actin and untagged P8
WH2 espin constructs. The microvilli examined at 4 h were selected to be of intermediate length (2.54.5 µm) to increase the likelihood that they were undergoing lengthening at the time of observation. Whether examined at 4 or 24 h, photobleached microvilli consistently showed a rapid (12 min) recovery of GFPß-actin fluorescence at their tips (Fig. 8 B). On the basis of the polarity of the filaments (Fig. 3), this was the location expected for filament barbed ends. The conclusion that filament barbed ends were concentrated at the microvillar tip and not elsewhere in the long microvillar PABs was supported by the pattern of rhodamine-actin incorporation observed in detergent-permeabilized cells. Rhodamine-actin fluorescence was highly concentrated at microvillar tips whether cells were labeled 4 h (Fig. 8, D and E) or 24 h (Fig. 8, F and G) after transfection with the
P8
WH2 espin construct.
After the rapid recovery of GFPß-actin fluorescence at the microvillar tip, the size of the recovered zone increased by extension toward the base of the microvillus at a relatively constant rate (Fig. 8 B), presumably because of actin treadmilling in the long microvillar PABs (Schneider et al., 2002; Tyska and Mooseker, 2002). The long microvilli frequently changed positions during monitoring and became obscured by other microvilli. This made it difficult to follow the extension of the zone of recovered fluorescence more than halfway down the microvillus. From measurements made at 2-min intervals during the first 610 min of recovery, the rates of movement of the recovered zone were 0.27 ± 0.01 µm/min (mean ± SEM; n = 138 microvilli, 20 cells) and 0.24 ± 0.01 µm/min (n = 105 microvilli, 17 cells) for microvilli examined 4 and 24 h after transfection, respectively. Assuming a rise per actin subunit of 2.75 nm in the F-actin helix, this suggested that the microvillar PABs in espin-expressing cells were undergoing actin treadmilling at a similar rate, 1.51.6 (actin subunits) s-1, during and after elongation.
We also examined the rate of actin treadmilling in the short microvilli of control cells 24 h after transfection with the GFPß-actin construct alone. The higher density of microvilli on control cells necessitated that the FRAP analysis be confined to subsets of microvilli in relative isolation near the edge of a given collection that were, because of irregularities in the monolayer, tipped more parallel to the substratum (Fig. 8 C). In addition, the shortness of the control microvilli reduced the number of time points that could be examined. For observations made during the first 46 min of recovery, the rate of movement of the recovered zone in the microvilli of control cells was 0.21 ± 0.01 µm/min (n = 143 microvilli, 19 cells), corresponding to an actin treadmilling rate of 1.3 s-1. Thus, espin did not slow actin treadmilling in microvillar PABs, but actually appeared to cause a slight increase.
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Discussion |
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More espin, longer PABs
The espins exerted a reproducible, concentration-dependent lengthening effect on the microvillar PABs of CL4 epithelial cells and could, presumably, have a lengthening effect on microvillus-type PABs in other cells, including the stereocilia of hair cells. At the highest espin level examined, microvillar PAB length increased an average of sixfold, but in extreme examples increased 13-fold. Significant 1.5- and 2.5-fold increases in length were observed at 2 and 10% of this maximum espin level, suggesting that supraphysiological levels are not required to observe an effect. Accordingly, the average lengths attained at the 2% (2.0 µm), 10% (3.4 µm), 40% (5.3 µm), and 100% (7.9 µm) espin levels span the range of lengths observed for the microvilli and stereocilia of most mammalian cells. Moreover, a similar correlation, in which a 910-fold increase in espin level is associated with a 2.53-fold increase in PAB length, was noted for the microvillar PABs of CL4 cells and the stereocilia of cochlear hair cells in situ.
Different actin-bundling proteins, different effects
Our results provide an illustration of how, in the same cell, different actin-bundling proteins become associated with, and exert their effects on, different classes of PAB-containing structures. Although espins became concentrated in the microvillar PABs and increased their length, fascin became concentrated in filopodium-like elements of protrusions that it caused to extend from the opposite end of the cell. This is consistent with the role proposed for fascin in the formation of filopodia (Svitkina et al., 2003) and suggests that the PABs of microvilli and filopodia differ qualitatively. The fimbrins are believed to contribute cross-links to the PABs of brush border microvilli and hair cell stereocilia (Mooseker, 1985; Tilney et al., 1989), and T-fimbrin accumulates in stereocilia during stereociliogenesis (Daudet and Lebart, 2002). Although T-fimbrin has been reported to cause some increases in microvillar dimensions in the LLC-PK1 parental line (Arpin et al., 1994), it was less active than the espins at increasing microvillar PAB length in CL4 cells. We uncovered an unanticipated functional similarity between espins and villin in their ability to increase the length of CL4 cell microvillar PABs. Our findings reinforce earlier work showing that villin can induce microvilli in transfected or microinjected cells (Franck et al., 1990; Friederich et al., 1992; Arpin et al., 1994) and are consistent with a role for villin in remodeling the enterocyte brush border after damage (Ferrary et al., 1999).
Elongation of existing microvillar PABs
Our results suggest that espins increase PAB length by causing a net barbed-end elongation of the treadmilling actin filaments that exist within the relatively short microvillar PABs of control CL4 cells before espin expression. Key support comes from the observation that the actin filaments of the long microvillar PABs in espin-expressing cells appear continuous from rootlet to tip. Although this continuity could arise secondarily, through additional nucleation events followed by a stitching together of loose ends, we saw no instances of bundle discontinuity, and the site of fluorescent actin incorporation (barbed ends) remained concentrated at the microvillar tip during and after lengthening. The existence of actin treadmilling in the long microvillar PABs, which we detected by FRAP, is further evidence in support of filament continuity. Also consistent with a mechanism involving the elongation of existing microvillar PABs is the inverse correlation we noted between microvillus number and thickness. Small espin tended to make microvilli that were relatively thin and more comparable in number to those found on control cells, whereas the larger espin isoforms made a smaller number of thicker microvilli. Thus, in addition to causing PAB elongation, the larger espin isoforms appear to cross-link the existing microvillar PABs to yield a smaller number of thicker microvilli. Finally, although we discovered that espins contain two types of functional domains commonly found in proteins involved in the nucleation of actin polymerization, namely an actin monomer-binding WH2 domain (Welch and Mullins, 2002) and profilin-binding proline-rich peptides (Evangelista et al., 2003), these two domains appear not to be required for PAB lengthening and are presumably required in other contexts. The net barbed-end elongation of treadmilling actin filaments in microvillar PABs mediated by espins may be one way that vertebrate cells make PABs longer without having to join bundle modules. Obvious espin orthologues are present in vertebrates from Fugu to human.
Effects of espins on actin polymerization/depolymerization reactions in PABs
The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for the microvillar PAB-lengthening effect. Moreover, lengthening required the two putative F-actinbinding sites disposed at opposite ends of the actin-bundling module. Thus, lengthening is likely attributable to espin cross-links. In view of espin's high affinity for binding and cross-linking F-actin in vitro (Chen et al., 1999), we were surprised that the microvillar PABs of the espin-expressing CL4 cells appeared to undergo actin treadmilling at a similar rate to those in control cells without espin. However, this was consistent with the modest effect of espin cross-links on actin depolymerization and treadmilling in vitro. Because espin can bind G-actin via its WH2 domain, the 1.31.9-fold decreases noted in the in vitro assays may actually be overestimates.
The rate of actin treadmilling we observed in the microvillar PABs of transiently transfected CL4 cells was 1.5 s-1. This is five times faster than the rate calculated by Tyska and Mooseker (2002) for the microvilli of control CL4 cells stably expressing GFPß-actin and closer to the rate in their partially differentiated cells (
3 s-1). It is also
20 and
150 times faster than the actin treadmilling reported for the stereocilia of transfected hair cells in cochlear explants isolated from postnatal day one to three rats and maintained in culture for 35 d or 1015 d, respectively (Schneider et al., 2002). Although 1.5 s-1 may be relatively fast for actin treadmilling in a microvillus-type PAB, it is 2.5 times slower than the average rate in stationary filopodia (Mallavarapu and Mitchison, 1999), and
550 times slower than the rates of actin filament turnover attained in the lamellipodia of migrating cells or in Listeria comet tails (for review see Pollard and Borisy, 2003).
The observation that the microvillar PABs of CL4 cells undergo actin treadmilling at 1.5 s-1 in the absence or presence of espin implies that espin cross-links cause PAB elongation through relatively subtle effects on the rates of actin polymerization and/or depolymerization. For example, when treadmilling is 1.5 s-1 (0.24 µm/min), the elongation of a PAB from 1.3 to 7.9 µm can be accomplished in 55 min by a transient twofold decrease in the rate of actin depolymerization at filament pointed ends or in 27.5 min by a transient twofold increase in the rate of actin polymerization at filament barbed ends. If 4 h are allotted, then elongation from 1.3 to 7.9 µm can be accomplished by a transient change in the rate of actin polymerization or depolymerization of
10%. Changes of such a small magnitude would be difficult to detect using in vitro assays and may depend on the contributions of other cellular proteins. Although the slight increase in actin treadmilling rate apparent in espin-expressing cells may be indicative of a subtle effect of espin on actin dynamics, an increase in treadmilling rate alone would be insufficient to cause lengthening.
Implications for the jerker phenotype
The microvillar PABs of espin-expressing cells do not elongate indefinitely, which suggests that the system regulates naturally to a steady-state. Moreover, the PABs continue to undergo actin treadmilling at 1.5 s-1 after reaching a steady-state length. Thus, the positive correlation between espin level and steady-state microvillar PAB length suggests that specific numbers of espin cross-links must remain present to balance actin depolymerization from filament pointed ends and maintain a stable length for the PAB. A requirement for espin to maintain the steady-state length of a treadmilling PAB could explain the shortening of hair cell stereocilia observed in homozygous jerker mice. The hair cells of these mice, which are deficient in espin protein because of a frameshift mutation in the espin gene (Zheng et al., 2000), have been reported to sprout and organize collections of stereocilia that appear relatively normal (Sjöström and Anniko, 1992). Although there has not yet been a quantitative analysis of stereocilium length in these mice, this suggests that espins are not required to establish a basic stereociliary array. Around postnatal day 10, the stereocilia of cochlear hair cells begin to collapse, shorten, and disappear in jerker homozygotes (Sjöström and Anniko, 1992). We hypothesize that around postnatal day 10 the PABs of stereocilia enter a new life-cycle phase characterized by faster actin treadmilling. This could reflect a physiological activation of the hair cell and might afford greater responsiveness to stimuli or facilitate repair. We propose that it is during this more dynamic phase that specific numbers of espin cross-links become necessary to balance a faster rate of actin depolymerization from filament pointed ends and maintain a fixed steady-state length for each stereocilium. On the basis of the weak activity of T-fimbrin in the microvillar PAB lengthening assay, the fimbrins of the stereocilium may not be able to compensate for the lack of espins. The seemingly stable and precise variations in PAB length maintained in the staircase array of stereocilia on each hair cell suggests that there may be mechanisms to compartmentalize and/or regulate espins locally within the hair cell.
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Materials and methods |
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CL4 cells were cultured at 37°C in minimum essential medium alpha medium (with L-glutamine, without nucleosides) supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. Cells cultured on glass coverslips for 10 d (75% confluency) were transfected for 4 h with a fixed amount of plasmid DNA using Lipofectamine (Chen et al., 1999) and examined 1724 h later. In some experiments, cytochalasin D (Sigma-Aldrich) was added after transfection from a 1,000-fold concentrated stock in DMSO. CL4 cells were fixed with PFA, treated briefly with 0.1% Triton X-100, labeled with antibodies and/or Texas redphalloidin (Molecular Probes) to detect F-actin, and mounted in 5% (wt/vol) n-propylgallate, 90% (vol/vol) glycerol (Chen et al., 1999). Transfected cells were identified by GFP fluorescence or, for untagged constructs or GFP constructs of low promoter strength, by immunofluorescence using the following antibodies with Alexa 488goat secondary antibodies (Molecular Probes): affinity purified rabbit polyclonal antirat Purkinje cell espin 1 (Sekerková et al., 2003), antirat espin COOH-terminal peptide (Zheng et al., 2000), mouse monoclonal anti-GFP (Roche), or antivillin (Immunotech). Cochlear whole mounts, obtained by dissection (Sobkowicz et al., 1993) of EDTA-decalcified bony labyrinths from PFA-fixed adult rats (Zheng et al., 2000), were labeled with affinity purified espin antibody and Alexa 488goat antirabbit secondary antibody and mounted in Vectashield (Vector) or labeled by the ABC immunoperoxidase procedure (Zheng et al., 2000).
0.5-µm confocal z-sections were collected at RT using a confocal microscope (model LSM 510 META; Carl Zeiss MicroImaging, Inc.) and a 100X, 1.4 NA oil immersion objective. LSM510 imaging software was used to generate orthogonal sections, from which measurements of microvillar length were made on cells selected at random from the 3040% of transfected cells that showed similar high levels of expression. Microvillar length measurements were made on those for which a complete course could be charted. Significance was evaluated by one-way ANOVA and confirmed using Dunnett's post-hoc test. Z-section images of hair cells from basal, middle, and apical turns of individual cochleas were collected using identical settings. Average intensity values for individual hair cells were measured from digitized confocal images using Metamorph (Universal Imaging Corp.). Images were saved in TIF format, transferred to Photoshop (Adobe Systems), assembled into composites and converted to CMYK color format with minor adjustments of brightness and contrast. CL4 cells were processed for EM without (Tilney et al., 1998) or with labeling with S1 (Svitkina and Borisy, 1998) and examined using an electron microscope (model JEM-1200 EX; JEOL). FRAP (Yoon et al., 1998) was performed on cells cotransfected with pEGFP-Cß-actin and pcDNA3 espin or
P8
WH2 espin. Filament barbed ends were localized by incorporation of rhodamine-actin after detergent permeabilization (Symons and Mitchison, 1991).
Western blotting was performed on hot SDS gel sample buffer extracts of replicate dishes of transfected cells using the ECL method (Amersham Biosciences). Rabbit skeletal muscle actin was polymerized and incubated with or without 6X His-tagged espin as described previously (Chen et al., 1999). 10 µM latrunculin A was added, and supernatants were collected by centrifugation for 20 min at 164,000 g in an ultracentrifuge (model TLA100.3 rotor/TL-100; Beckman Coulter) and analyzed in Coomassie bluestained SDS gels. Actin depolymerization/treadmilling was assayed using the sedimentation version of the method of Carlier et al. (1997), in which [2,8-3H]ATP (Amersham Biosciences) was substituted for fluorescent ATP analogue. The chase with unlabeled ATP was started 60 min after adding 6X His-tagged espin or buffer. Supernatants were collected by centrifugation (see above), and the released 3H-adenine nucleotide was measured in a scintillation counter. In some experiments, 3 µM human cofilin was added 20 min before the chase. GST pull-down assays were conducted as described using either 6X His-tagged espin constructs (Sekerková et al., 2003) or G-actin (Uruno et al., 2001) as ligand.
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Acknowledgments |
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This work used the confocal in the Northwestern University Cell Imaging Facility. We are especially indebted to B. Fritsch and K. Beisel for suggesting that we search for differences in espin protein level among hair cells. This work was supported by grant DC004314 to J.R. Bartles from the National Institute on Deafness and Other Communication Disorders.
Submitted: 15 September 2003
Accepted: 27 October 2003
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