1Department of Medicine, Indiana University School of Medicine, and 2Department of Biology, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana
Submitted 15 December 2003 ; accepted in final form 11 February 2005
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ABSTRACT |
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actin; cytoskeleton; ion channel; kidney
Modulation of actin structure and actin-dependent processes is often mediated via members of the myosin family of proteins. Myosin I proteins contain ATP-, actin-, and membrane-binding domains that make them excellent candidates to regulate exocytotic and endocytotic events as well as to serve as participants in the regulation of integral membrane proteins such as channels (2). Support for an active role of myosin I and other myosins in these processes exists in multiple systems (12, 15, 17, 49).
We previously identified (9) Myo1c, previously called myosin I or Myr2, as a myosin I enriched in the brush border of proximal tubule cells. After an ischemic insult that caused the collapse of the actin-rich brush border, Myo1c was found in cellular blebs and urine. Its return to the brush border, as observed by immunofluorescence, occurred after the structural recovery of the actin in the brush border. Interestingly, Myo1c was observed to be present in most, if not all, kidney tubules, including the collecting duct.
The ubiquitous distribution of Myo1c suggests that it likely participates in fundamental cell functions. Research from several laboratories supports the contention that Myo1c is the best candidate for the adaptation motor of hair cells (21). This process depends on interactions between actin and an ion channel located in the apical portion of stereocilia. A more recent study suggests that depletion of Myo1c from adipocytes, with small interfering RNA, reduces the transport of Glut4-containing vesicles to the plasma membrane (7). These studies implicate Myo1c in actin-associated functions at the cortex of various cell types.
The kidney collecting duct's primary function is to make final adjustment of urinary solute osmolarity and concentrations (43). These tubules consist of two functionally distinct epithelial cellsthe principal and intercalated cells. The principal cells respond to antidiuretic hormone (ADH) or vasopressin by transporting aquaporin channels to the apical membrane. Vasopressin also stimulates the epithelial Na+ channel ENaC, present in principal cells. Data from several laboratories support the idea that ENaC is inserted from a cytoplasmic pool after ADH stimulation (5, 18, 48). In addition, a role for the cortical actin cytoskeleton has also been shown. Cl channels including CFTR are also present in both principal and intercalated cells and in M1 cells (30, 31, 45, 53). The mechanism(s) that is used to regulate CFTR's Cl transport and its interactions with other channels including ENaC is complex and involves both trafficking and regulatory components (4).
By immunofluorescence, Myo1c is seen to be concentrated at the plasma membrane in M1 cells. We hypothesized that ENaC channel insertion depends on actin-myosin interactions and that disruption of Myo1c would adversely affect the normal ion transport response after ADH stimulation of M1 cells. To test our hypothesis, we created a truncated Myo1c lacking both ATP and actin domains and stably expressed this construct in M1 cells. Evaluation of ion transport responses is consistent with Myo1c having a role in ENaC activation via channel delivery and/or insertion into the plasma membrane.
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MATERIALS AND METHODS |
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Cell culture. The M1 cell line, originally developed by Dr. G. Fejes-Toth (50), was obtained from the American Type Culture Collection (Manassas, VA). Cultures were routinely grown on plastic in DMEM-Ham's F-12 medium with L-glutamine and 15 mM HEPES supplemented with 2.5% Cosmic calf serum (HyClone, Logan, UT), 2.5% fetal bovine serum, 100 U penicillin, and 100 µg/ml streptomycin. Costar Transwell polycarbonate filters, 0.4-µm pore size, were used for electrophysiological assays and some immunofluorescence experiments. Cells were used on days 2022 after seeding onto filters (2 x 105 cells/cm2) for Ussing chamber experiments.
Construction of Myo1c constructs.
Standard molecular techniques were used to create a truncated Myo1c construct, which was inserted into the pcDNA6/V5-His plasmid (Invitrogen, Carlsbad, CA). PCR was used to amplify a Myo1c tail construct that contained all the IQ motifs (start nucleotide 2289). The template DNA was the full-length rat Myr2 construct from Dr. Martin Bahler, University of Münster, Germany (42), and the two primers used were the following: forward 5'-CTGGATCCATGGGCAGGACTAAGATC-3' and reverse 5'-AGGACACCTAGTCAGACAAAATGATGC-3'. The forward primer contained a BamHI site and the 3' end of the construct an EcoRI site that were subsequently cut to enable ligation with pcDNA6/V5-His plasmid cut with BamHI-EcoRI. Proper placement was verified by DNA sequencing of the Myo1c IQ tail pcDNA6/V5-His construct (Myo1cIQ). Similar methods were used to place the full-length Myr2 construct into pcDNA6/V5-His plasmid. M1 cells were transfected with Myo1cIQ, Myo1cFL, or pcDNA6/V5 lacking any insert with Lipofectamine (Life Technologies, Rockville, MD), and expressing cells were selected with blasticidin (10 µg/ml). Two rounds of selection were performed with cloning rings. M1 cells stably expressing the Myo1c IQ construct (M1IQ) or Myo1cFL construct (MF3) are maintained in blasticidin (4 µg/ml) containing medium, and express the truncated Myo1c at levels of 25% of the endogenous Myo1c, based on immunoblot. Identification of the Myo1c constructs was accomplished by Western blotting with either Myo1c or V5 antibodies. We have been unable to label the Myo1c constructs by immunofluorescence with either V5 or His antibodies. Construction of enhanced green fluorescent protein (EGFP)-Myo1c plasmids also used the full-length Myr2 construct as starting template to place the full-length, IQ (lacking ATP- and actin-binding domains), and Tail (lacking ATP-, actin-, and first 3 IQ domains; Ref. 16) Myr2 constructs into the appropriate version of pEGFP-C from Clontech (Palo Alto, CA). Briefly, full-length Myr2 was cut with EcoRI and ligated into pEGFP-C2 cut with EcoRI. With similar methods Myo1c constructs lacking ATP- and actin-binding domains, IQ-EGFP (start nucleotide 2289), and T-EGFP (start nucleotide 2595), which lacks ATP, actin, and IQ motifs, were constructed. EGFP for all three constructs was placed on the amino end and confirmed by sequencing. A diagram of these constructs and their respective domains is presented in Fig. 1.
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Triton X-100 extraction of cells was performed with the following buffer (in mM): 50 imidazole, 50 KCl, 0.5 MgCl2, 0.1 EDTA, 1 EGTA, and 1 DTT with 1% Triton X-100 and protease, kinase, and phosphatase inhibitors. Cells on filters were rapidly cooled, scraped, and pelleted in cold Krebs buffer. The pellet was extracted for 1015 min on ice with the above buffer, followed by centrifugation for 30 min at 100,000 g in a Beckman table top ultracentrifuge (rotor 120.1). Pellets and supernatants were made equal volume with sample buffer and analyzed by Western blotting. Quantitation of Western blots was performed with UN-SCAN-IT (Silk Scientific, Orem, UT) digitizing software.
Immunofluorescence. Fixation and immunolabeling of kidney sections was performed as described previously (9). Briefly, rat kidneys were perfused fixed according to the procedure of Maunsbach and Afzelius (36). Fifty- to one hundred-micrometer sections were obtained with a vibratome. Tissue was permeabilized, with either 1% SDS or 1% Triton X-100, for 5 min, followed by antibody labeling. Myo1c primary antibodies were used (510 µg/ml), and secondary antibodies were conjugated to Cy5 (Jackson ImmunoResearch) or Alexa dyes (Molecular Probes). F-actin was labeled by including Alexa 488 or Oregon Green phalloidin (Molecular Probes) diluted 1:200 with the secondary antibody.
Glass- or filter-grown cells were fixed with 24% paraformaldehyde and permeabilized with either 0.5% Triton X-100 or 0.05% saponin in PBS. Immunolabeling for all antibodies was performed as previously described, using the Myo1c antibodies (54, 55).
Transient transfection. Cells were plated onto Mattek (Ashland, MA) coverslip dishes and transfected with either Effectene (Qiagen, Valencia, CA) or Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Transfected cells were imaged live with the Zeiss LSM 510 confocal microscope and a x60, 1.4-numerical aperture (NA) water objective. Temperature and pH were controlled by use of a Warner warm stage holder and placement of cells into a HEPES-Krebs buffer (in mM: 1 MgCl2, 1 CaCl2, 55 NaCl, 3 KCl, 10 dextrose, 10 HEPES, and 150 sucrose).
Microscopy. Images were collected with either a MRC-1024 laser scanning confocal microscope (Bio-Rad) or a Zeiss LSM 510 confocal microscope at the Indiana Center for Biological Microscopy. In both cases, high-NA objectives were used. Z stacks were collected with a step size between 0.2 and 0.5 µm. Selected images were imported into Adobe Photoshop and labeled with Adobe Illustrator.
Electrophysiological studies. Cells were grown for 2022 days on 2.5-cm Falcon Transwell polycarbonate filters or Snapwells. The filters were clamped between the halves of an Ussing chamber (World Precision Instruments, Sarasota, FL) (30). The fluid chamber was maintained at 37°C with a water-jacketed buffer reservoir. pH was maintained and buffer circulated with a 5% CO2-95% O2 gas lift. The electrodes were connected to a voltage-clamp amplifier (current voltage clamp; World Precision Instruments) for measurement of net ion flux as monitored under short-circuit conditions [short-circuit current (SCC)] (28). Transepithelial resistance was calculated by intermittently applying a 2-mV pulse across the epithelium and measuring the resultant deflection in SCC.
Serum-free DMEM-F-12 medium was used for studies shown in Figs. 4 6. Filters were placed into the chambers and incubated under SCC conditions until a steady baseline was obtained (0.51 h). Concentrations and times of addition of the various compounds are indicated in Figs. 46. Each experiment was performed with matched cultures grown in parallel.
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RESULTS |
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The first question addressed with the M1 cells was Myo1c localization and the effect of expression of the Myo1c IQ construct on endogenous Myo1c's distribution and F-actin structure. Figure 3 shows the location of Myo1c in M1 and M1IQ cells grown on filters. Both x-y planes and x-z cross sections (apical membrane at top) are presented. X-z images show only Myo1c location. The concentration of Myo1c at the apical and lateral membranes is seen in both M1 and M1IQ cells. The arrows in x-z sections point to areas containing a high concentration of Myo1c, which may correspond to special membrane/cytoskeletal domains. No significant difference was observed in Myo1c location or the actin cytoskeleton in parental M1 cells or M1IQ cells expressing the truncated Myo1c construct. The presence of Myo1c near the plasma membrane, often in distinct domains, is consistent with this myosin having a role in membrane events such as channel regulation. Myo1c location in M1C and MF3 cells was comparable to that in M1 and M1IQ cells (data not shown).
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Figure 4 shows the response for both the parental M1 cells and the M1IQ cells that contain both endogenous Myo1c and truncated Myo1c lacking ATP and actin domains. There was a diminished response of the M1IQ cells to ADH and an apparent reduction of amiloride-sensitive transport in these cells. These results are consistent with a reduced delivery and/or activation of ENaC. The subsequent addition of DBcAMP resulted in an amplified response for the M1IQ cells in the presence of the Na+ channel inhibitor amiloride. The amplified DBcAMP response in the M1IQ cells suggests that a Cl channel may be affected. A reciprocal activation arrangement between ENaC and CFTR has been observed in M1 cells (31) and other cell types (35, 45). Differences between cell types were statistically significant.
To further substantiate the participation of Myo1c in the ADH/Na+ channel response, we created M1 cells expressing the full-length Myo1c (MF3). Recent data suggest that overexpression of Myo1c promotes membrane fusion (8); thus these cells may have increased delivery of membrane proteins including channels. In Fig. 5A, M1 and M1IQ cells were analyzed, along with MF3 cells, using electrophysiological techniques on confluent monolayers. As in Fig. 4, there was a diminished ADH response, minimal to no amiloride effect, and increased cAMP response in the M1IQ cells compared with the M1 parent line. Interestingly, MF3 cells have a significantly increased basal SCC that is also not responsive to ADH or amiloride but responds to cAMP like the M1IQ cells. Thus whether truncated Myo1c is expressed or increased expression of full-length Myo1c occurs, a diminished ADH response and much-reduced amiloride inhibition are observed. The increase in SCC observed in the MF3 cells may be the result of increased channel density or activation. In Fig. 5B M1 cells expressing the empty pcDNA6/V5-His plasmid (M1C) were compared with M1 cells, and no significant differences were observed. The average peak ADH response was 8 µA/cm2 for M1 cells and 5.9 µA/cm2 for M1C cells. The average peak cAMP response was 9.1 µA/cm2 for M1 cells and 7.1 µA/cm2 for M1C cells. As in Fig. 5A these cells were run on the same day under identical conditions, using cells grown in parallel.
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To begin characterization of the increase in ion transport observed after DBcAMP addition, we evaluated the effects of three Cl channel inhibitors on the DBcAMP response in the M1IQ cells (23, 26, 37, 44). These studies used a defined bath solution on both the apical and basolateral sides of the cells (in mM: 140 Na+, 4 K+, 1 Ca2+, 1 Mg2+, 124 Cl, 24 HCO3, and 5 D-glucose) (24). The Cl channels targeted were CFTR, swelling-activated Cl1 (ICl,swell), and Ca2+-activated Cl. Niflumic acid has a higher affinity for the Ca2+-activated Cl channel than either the ICl,swell or CFTR channels, whereas tamoxifen is classified as a ICl,swell channel inhibitor (44). Neither niflumic acid (50 µM) nor tamoxifen (50 µM) inhibited DBcAMP-stimulated current increase in the presence of amiloride (data not shown). NPPB (100 µM) completely inhibited the response. The inhibition by NPPB supports a role for Cl channels and, combined with the other inhibitor results, points to CFTR, whose presence in these cells is suggested by electrophysiology studies (31) and was shown by RT-PCR experiments (53). Additional evidence that Cl transport accounts for the SCC after DBcAMP addition came from studies using low-Cl buffers that showed an absent or much-reduced SCC change after DBcAMP (data not shown).
Measurement of resistances in the M1 parent line (1,134 ± 58 /cm2; n > 10) vs. MC1, M1IQ, and MF3 cells showed that M1 cells and MC1 cells have essentially identical resistances. In contrast, the M1IQ cell resistance was more variable, always less than one-half of that of M1 and most often
220
/cm2. The MF3 cells had the lowest resistance, which was 34 ± 24
/cm2 (n > 10).
Does expression of truncated Myo1c alter actin association as monitored by Triton X-100 extraction? Several studies suggest that myosin I can self-associate, possibly through tail-tail interactions (20, 38, 55). Given the similarity in Myo1c immunofluorescent staining in M1 and M1IQ cells, observed by using the Myo1c M2 antibody that recognizes both full-length and IQ truncated forms, it is reasonable to propose that the truncated form incorporates into endogenous Myo1c complexes. In these experiments three questions were addressed. First, does the incorporation of truncated Myo1c alter the Triton X-100 extractability of endogenous Myo1c? Second, does addition of DBcAMP alter Myo1c Triton X-100 extraction properties? Third, is the truncated Myo1c construct extracted with Triton X-100?
Filter-grown cells were extracted under control conditions and after addition of 1 mM DBcAMP for 2 min. Figure 7 shows the results from Western blots that were scanned and quantitated. The total quantity of Myo1c (pellet + supernatant) was set at 100%, and the percentage present in the supernatant, Triton soluble, is shown in the absence (Fig. 7, A, C, and E) and presence (Fig. 7, B, D, and F) of DBcAMP. There was a significant shift of Myo1c to increased solubility when the truncated Myo1c IQ construct was present (compare Fig. 7A with Fig. 7C; P = 0.021, unpaired Student's t-test). The addition of DBcAMP to control M1 cells also triggered an apparent rearrangement that resulted in more Myo1c being Triton X-100 extractable (compare Fig. 7A with Fig. 7B). The variability between M1 filters is greater than in the M1IQ cells, and the P value by paired Student's t-test comparing Fig. 7A to Fig. 7B was 0.056. No difference was observed between the M1IQ cells after DBcAMP addition (compare Fig. 7C with Fig. 7D). Figure 7, E and F, show that the truncated Myo1c construct is 90% Triton X-100 soluble, with the addition of DBcAMP causing no change.
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DISCUSSION |
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In these studies we have addressed the function of an unconventional myosin I, Myo1c, in collecting duct cells. The model used was the well-established M1 mouse cortical collecting duct cell line. Our working hypothesis was that ENaC channel insertion is dependent on actin-Myo1c interactions. The delivery and insertion of ENaC channels to the plasma membrane after ADH stimulation is well established (18, 48). In contrast, ADH and subsequent cAMP increase is thought to cause both the activation of existing CFTR plasma membrane channels and the insertion of new channels (4). Our approach compared the responses of parental cells to those of cells stably expressing a truncated, motor domain-deleted Myo1c and cells overexpressing Myo1c. Myo1c, as well as the truncated construct, was found to be concentrated at the plasma membrane, often in discrete domains. Expression of truncated Myo1c modulated the ADH response, and these cells had a diminished amiloride inhibition. Surprisingly, expression of the full-length construct also resulted in an altered ADH response and diminished amiloride inhibition. These results are consistent with these cells having diminished ENaC plasma membrane delivery and or activation. The observed increase in SCC after DBcAMP addition likely represents Cl movement, possibly via CFTR. Understanding this change that occurs in the presence of amiloride will require further investigation. The M1IQ cell alterations observed may be the result of incorporation of truncated Myo1c into endogenous Myo1c complexes. The truncated construct would be unable to perform any mechanochemical function and might also disrupt Myo1c's normal protein or lipid associations that occur during activation of channels. The MF3 cells, which overexpress Myo1c, have a much higher basal SCC in addition to ADH and amiloride responses similar to the M1IQ cells. The two most likely explanations for the high basal SCC are increased channel density and increased activation of channels. In either case, the overexpression of Myo1c appears to have resulted in altered channel regulation whereby some channels are preferentially inserted or active at the plasma membrane while the ADH-responsive Na+ channel is reduced. Deciphering which channel(s) is responsible for the MF3 cell basal SCC will contribute to a better understanding of how Myo1c contributes to the complex cytoskeleton regulation of channel activity.
The identification of Myo1c binding partners has been difficult, with recent studies suggesting that the IQ domain is important (16). In fact, in expressed Myo1c fragments, Gillespie's results (16) in hair cells suggest that the second IQ domain must release calmodulin before receptor binding. Our studies with EGFP-Myo1c constructs show that the EGFP-Myo1cIQ construct does, indeed, colocalize to the same cell regions along the plasma membrane as the full-length construct. However, the EGFP-tail construct that lacks both the motor and IQ domains is present in a diffuse cytoplasmic pattern. Also, in agreement with our immunofluorescence results, none of the EGFP-Myo1c constructs localized to any actin stress fibers. Myo1c may be selectively targeted to specific actin areas by actin-associated proteins, such as tropomyosin, that prevent or possibly promote Myo1c actin binding.
The effect of calcium on myosin I's calmodulin and phospholipid interactions was first studied with brush border myosin I. Swanljung-Collins and Collins (51) proposed a calmodulin/phosphatidylserine switch whereby at micromolar calcium concentrations a calmodulin was released with an increase in lipid binding. A more recent study using expressed Myo1c tail protein suggested that calcium decreases Myo1c lipid association (52). It is also clear that both Myo1c's actin motility and actin-dependent ATPase activity are affected by calcium (3, 60). The regulation of Myo1c is likely to have both common and unique parameters that are dictated by cell type. In the collecting duct, one possibility is that on cell stimulation a local calcium increase causes the release of one calmodulin molecule from Myo1c, thus revealing a new protein-binding site. Another possibility may involve a change in the IQ domain conformation resulting from lipid binding to the tail domain. The release of calmodulin from Myo1c may also contribute to the regulation of other calmodulin-binding proteins. Where and when Myo1c releases and binds calmodulin are important questions that require further investigation.
Myo1c regulation of a channel or channel effector complex near the plasma membrane would likely involve movement or rearrangement of the cortical actin cytoskeleton. Two possible mechanisms by which Myo1c could alter ion channel properties include altering channel insertion into plasma membrane and altering the cortical actin cytoskeleton, which indirectly affects the channel's properties. Actin-dependent motility would follow calmodulin release and receptor-cargo binding initiating delivery of cargo to membrane domains or modulation of cortical actin cytoskeleton, thus resulting in full activation of the channel. Alternatively, Myo1c actions may result in inhibition of activity by preventing necessary protein or lipid interactions. Phosphorylation of Myo1c will likely have a role in its activity and interactions. Regulation of cargo interaction by phosphorylation may be a common mechanism used by molecular motors (27, 48). It is possible that a phosphorylation event on Myo1c serves as the brake or signal once the necessary cortical cytoskeleton or membrane movement has occurred. The kinase and phosphatase that are responsible may be spatially restricted, thus contributing to the regulation of these events. The cessation of myosin actin-based motility may also have a filament-based component, as actin bound with tropomyosin significantly reduces myosin I-based activity and motility (19).
A recurring theme involving complex signaling pathways is establishment of specific domains or assemblies of proteins that raise the local concentration of each protein and any signaling intermediates that may be generated (23). This not only reduces the distance needed for communication but enables faster responses and allows multiple signaling events to take place with less cross talk. Detergent-resistant membranes, i.e., lipid rafts and caveolae, can serve as assembly sites for channels and effectors, thus facilitating interactions and activity (47, 59). Interestingly, ENaC and Myo1c both associate with anionic phospholipids (34, 52).
These results, combined with the results of earlier studies, provide compelling evidence that Myo1c is an important actin-dependent motor protein that participates in the modulation of ENaC in M1 cells. M1 cells expressing a truncated Myo1c, lacking ATP- and actin-binding domains, have altered SCC when stimulated with ADH, which has been shown to result in ENaC insertion into the plasma membrane. Ion channel function depends on insertion and removal of channels from the plasma membrane in addition to specific activation signals. Many of these events take place near or within the cortical actin cytoskeleton, which has been implicated in controlling a diverse array of channels. Our hypothesis is that Myo1c, through its interactions with actin, membranes and membrane binding proteins, and calmodulin is a key regulator of these events for ENaC. Live cell imaging of fluorescently tagged channels in cells expressing Myo1c mutations will help to clarify Myo1c's contribution to channel function.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of A. Srirangam: Dept. of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202.
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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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