The role of TMEM16A (ANO1) and TMEM16F (ANO6) in cell migration
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Members of the TMEM16 family have recently been described as Ca2+-activated Cl− channels. They have been implicated in cancer and appear to be associated with poor patient prognosis. Here, we investigate the role of TMEM16 channels in cell migration, which is largely unknown. We focused on TMEM16A and TMEM16F channels that have the highest expression of TMEM16 channels in Ehrlich Lettre ascites (ELA) cells. Due to the lack of specific pharmacological modulators, we employed a miRNA approach and stably knocked down the expression of TMEM16A and TMEM16F channels, respectively. Migration analysis shows that TMEM16A KD clones are affected in their directional migration, whereas TMEM16F KD clones show a 40 % reduced rate of cell migration. Moreover, TMEM16A KD clones have a smaller projected cell area, and they are rounder than TMEM16F KD clones. The morphological changes are linearly correlated with the directionality of cells. TMEM16A and TMEM16F, thus, have an important function in cell migration—TMEM16A in directional migration, TMEM16F in determination of the speed of migration. We conclude that TMEM16A and TMEM16F channels have a distinct impact on the steering and motor mechanisms of migrating ELA cells.
KeywordsANO1 TMEM16A ANO6 TMEM16F CaCC Migration
Cell migration is involved in important physiological and pathophysiological processes ranging from embryogenesis to tumor metastases. On a cellular level, it is a process that involves the precise temporal and spatial integration of many components of the cellular migration machinery. One of these components, whose crucial role was appreciated only in recent years, is comprised of ion transport proteins. Numerous ion channels and transporters have been shown to be required for efficient migration of many different cell types (see [31, 32, 36] for review). Several general mechanisms have been identified, by which ion transport proteins impact on cell migration. They include but are not limited to the regulation of the cell volume and of the intracellular Ca2+ concentration ([Ca2+]i).
Many of the ion transport proteins that are part of the cellular migration machinery are involved in the regulation of the cell volume . Based on this observation, we proposed a model that comprises local swelling at the cell front during the protrusion of the lamellipodium and local cell shrinkage at the rear part of the cell during its retraction . By and large, this model was confirmed experimentally by showing (local) volume changes of migrating cells of up to ∼30 % [12, 27, 38] and by revealing the functional expression of the respective transport proteins such as KCa3.1 channels, NHE1, NKCC1, or AQP1 either at the cell front or at the rear part of migrating cells [11, 18, 25, 33]. In order to induce local shrinkage, KCa3.1 channels need to cooperate with anion channels. In glioblastoma cells, ClC3 channels were proposed to constitute the major route for the required anion efflux [5, 21]. Since ClC3 indirectly acquires Ca2+ sensitivity via its interaction with Ca2+/calmodulin-dependent protein kinase II, its activity can be coordinated with that of KCa channels and other Ca2+ dependent cytoskeletal mechanisms of cell migration. Alternatively, volume-regulated anion channels were proposed to cooperate with KCa3.1 channels . Cell migration is a Ca2+-dependent process, and loading cells with the Ca2+ chelator BAPTA inhibits migration . Thus, it is also likely that Ca2+-activated Cl− channels could be involved.
Recently, TMEM16A and B also called anoctamin 1 and 2 (or ANO 1 and 2) were identified as molecular correlates of Ca2+ sensitive Cl− channels [4, 6, 29, 40]. TMEM16F or ANO6 was also recently shown to be a Ca2+-activated Cl− channel with delayed Ca2+ activation [10, 35]. To date, ten members of the TMEM16 gene family have been identified in mammals (TMEM16A-K (I is omitted) or ANO1–10). They are conserved throughout the eukaryotic kingdom and contain eight-pass transmembrane segments with the N and C termini located in the cytosol and a reentrant loop situated between TM5 and TM6 possibly comprising the pore domain [9, 13, 14]. Each member is predicted to have multiple isoforms being simultaneously expressed in a given cell . Nonetheless, the TMEM16 family shows tissue-specific expression. mRNA abundance of all members has been assayed in murine tissue, showing that TMEM16A, F, G, H, J, and K (ANO1, -6, -7, -8, -9, and -10) are expressed in various epithelial tissues, while TMEM16B, C, D, and E (ANO2, -3, -4, and -5) are primarily expressed in neuronal and musculoskeletal tissues . One study has shown that members of the TMEM16 family (especially TMEM16A, F, and K) often are expressed in the same tissue . This observation could indicate that the family members work in concert compensating for each other upon disarrangements.
In the context of the present paper, it is noteworthy that TMEM16/ANO proteins have been known for some time to be upregulated in cancer and to be associated with poor patient prognosis . Moreover, some studies pointed towards a role of TMEM16A/ANO1 channels in tumor cell migration [2, 24]. In the present study, we tested whether other TMEM16 isoforms also contribute to migration of Ehrlich Lettre ascites (ELA) cells, and whether they can compensate each other.
Materials and methods
Cell culture and generation of mTMEM16A and mTMEM16F stable knock-down Ehrlich Lettre ascites cell lines
Experiments were performed using ELA cells. Cells were kept at 37 °C with 5 % CO2:95 % air and cultured in RPMI1640, with 10 % fetal bovine serum and 1 % penicillin/streptomycin added to the media. Cells were transfected with miR-16A-KD plasmid or miR-16F-KD (see construction of microRNA plasmids) plasmid using Lipofectamine 2000 (Invitrogen, Taastrup, Denmark) and incubated for 4 h, then resuspended in selection medium containing blasticidin (10 μg/ml). After 1 week of selection, single cells were picked and transferred to 96-well trays and allowed to grow into a full clone in the presence of blasticidin. Clones were screened for knockdown using qPCR and Western blot analysis. The selected clones were named TMEM16A-5, TMEM16A-7, TMEM16A-9, TMEM16F-10, TMEM16F-15, TMEM16F-16, and TMEM16K.
Construction of microRNA plasmids
Interfering microRNA (miRNA) KD of mTMEM16A and mTMEM16F was generated using BLOCK-iT™ Pol II miR RNAi expression vector kits (Invitrogen, Taastrup, Denmark). miRNA targeting mouse TMEM16A and TMEM16F were designed using the Invitrogen® online design tool generating sense and antisense single stranded DNA strings (ssDNA). DNA sequences were; TMEM16A-5 and 7: sense 5'-tgctggtaacttgcccattcctcatagttttggccactgactgactatgaggagggcaagttac-3', TMEM16A-5 and 7: antisense 5 -cctggtaacttgccctcctcatagtcagtcagtggccaaaactatgaggaatgggcaagttacc-3', TMEM16A-9: sense 5'-tgctgtttggaggaagccgagtagtcgttttggccactgactgacgactactccttcctccaaa-3', TMEM16A-9: antisense 5'-cctgtttggaggaaggagtagtcgtcagtcagtggccaaaacgactactcggcttcctccaaac-3', TMEM16F: sense 5'-tgctgtttagcgggagtttgatgtgcgttttggccactgactgacgcacatcactcccgctaaa-3', TMEM16F: antisense: 5'-cctgtttagcgggagtgatgtgcgtcagtcagtggccaaaacgcacatcaaactcccgctaaac-3', TMEM16K: sense 5'-tgctgagcatcagggctaagttcagggttttggccactgactgaccctgaactgccctgatgct-3', and TMEM16K: antisense 5'-cctgagcatcagggcagttcagggtcagtcagtggccaaaaccctgaacttagccctgatgctc-3'.
The two ssDNAs were annealed generating a dsDNA, which were then ligated into pcDNA™6.2-GW/EmGFP-miR generating miR-16A-KD, miR-16 F-KD, and miR-16 K-KD. Plasmids were transformed into OmniMax T1 Escherichia coli (Invitrogen, Taastrup, Denmark) and selected using spectinomycin as antibiotic (50 μg/μl). Plasmid inserts were confirmed by DNA sequencing.
Isolation of RNA, cDNA, and qPCR
Where Etarget and Eref are the amplification efficiencies for the target gene and the average of the two reference genes ARP and mB2m, respectively. ΔC(t)target and ΔC(t)ref are the change in C(t) values for target and reference genes.
Gel electrophoresis and Western blotting
Cells were lysed in 95 °C lysis buffer (150 mM NaCl, 20 mM HEPES, 1 mM EDTA, 10 % Glycerol, 0.5 % Triton X-100, and 0.5 % SDS) with protease inhibitors (Roche Applied Science) and phosphatase inhibitors added. The lysates were subsequently homogenized, and the supernatant was collected by centrifugation. Protein concentrations were determined using Bio-Rad DC protein assay, and identical amounts of protein was added to each lane. SDS-polyacrylamide gel electrophoresis was performed using a 10 % gel (Invitrogen), and size-fractionated proteins were transferred to nitrocellulose membranes. The membranes were stained with Ponceau Red (Sigma) as a second control of identical protein loading in each lane and washed in TBST (10 mM Tris HCl, pH 7.5, 120 mM NaCl, and 0.1 % Tween 20) before being blocked in a 5 % nonfat dry milk and TBST solution. Membranes were incubated with primary antibody at following concentrations: TMEM16A; ab53212 (abcam) 1:50, TMEM16A; Ano1 AB (DOG1AB) 1:500; TMEM16F; sc-136932 contingent (Santa Cruz) 1:250, β-actin; A1978 (Sigma) 1:1,000. Membranes were washed in TBST before incubation with the secondary antibody (Sigma) 1:5,000. The protein bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (Kirkegaard and Perry Lab) and scanned.
Cells were grown on poly-L-lysin-coated coverslips. Whole-cell patch-clamp recordings were performed using the Axopatch 200B amplifier interfaced to a Digidata 1440A controlled by pClamp10 software (Molecular Devices). Analog signals were acquired at 2.5 kHz and filtered at 1 kHz. Patch electrodes were pulled from borosilicate glass and had an input resistance of 2.5–6 MΩ. All recordings were performed at room temperature (20 °C). The standard bath solution contained (in millimolar): 150 NaCl, 1.5 CaCl2, 1 MgCl2, 10 Glucose, 10 HEPES, pH 7.4, and osmolality 315 mOsm. The 1 μM free Ca2+ pipet solution contained (in millimolar): 100 CsAsp, 40 CsCl, 4.34 CaCl2, 4 Na2ATP, 1 MgCl2, 5 EGTA, 10 HEPES, pH 7.2, and osmolality 295 mOsm.
Migration of ELA wild-type (wt) and ELA KD cells was assessed with a wound-healing assay. When the cell monolayers were confluent, a scratch was made with a fine pipette (width ∼260 μm). Then, the culture medium was replaced with serum-free medium, and the cells were allowed to recover in the incubator for 1 h. The tissue culture flasks were thereafter sealed and placed in a heating chamber (37 °C) on the stage of an inverted microscope (Axiovert 25 or Axiovert 40C; ×10 or ×20; Zeiss, Oberkochen, Germany). Migration was monitored with a video camera for 4 h (Hamamatsu, Herrsching, Germany) and controlled by HiPic software (Hamamatsu). Images were analyzed in 15-min intervals by marking the outlines of individual cells semiautomatically at each time step with Amira software (TGS Inc., San Diego, CA, USA). These segmentation data were used for further processing. Migration was quantified as the movement of the cell center (geometric mean of the pixel positions within the cell outlines) and the rate determined as the displacement of the cell center with time (micrometer per minute). Translocation (micrometer) is the distance between the position of cells at the beginning and at the end of the experiment. The trajectories of individual cells were normalized to a common starting point (t = 0 h). The cell area (square micrometers) is the appropriately scaled sum of all pixels inside the cell contours in two dimensions. The structural index (SI = 4π A/p 2) reflects the “roundness” of a cell with A representing the cell area and p the cell perimeter. It is one for a circle and approaches zero for a very elongated cell. The directionality of cell migration during wound closure was assessed by calculating the velocity of migration perpendicular to the wound (micrometer per minute). The directionality index is the ratio of translocation and total path length. We analyzed at least n = 30 cells from ≥N = 3 experiments for each condition.
All data are presented as representative original experiments or as means ± standard error of the mean (SEM). The statistical significance of differences was assessed with paired or unpaired Student's t test as appropriate. miTMEM16 clones were compared with the mock transfected clone. The level of significance was set to p < 0.05.
Verification of TMEM16A and TMEM16F knockdown in Ehrlich Lettre ascites cells
Using qPCR, we analyzed the mRNA expression levels of TMEM16A, 16 F, and 16 K in all cell lines (Fig. 1b). All miTMEM16A clones had a reduced TMEM16A mRNA expression level ranging between 50 (clone 5) and 80 % (clone 9) of the control level. However, the TMEM16F mRNA level was elevated to 180 % of the control level in miTMEM16A-5 clone. Similarly, there was a trend towards elevated TMEM16F expression in miTMEM16A-7 and -9 clones. TMEM16K expression level was not affected by the knockdown of TMEM16A (Fig. 1b). All selected miTMEM16F clones showed a reduced TMEM16F mRNA level. Notably, two of the three clones (miTMEM16F-10 and -16) also had reduced TMEM16A mRNA levels, ranging between 50 and 80 % of the respective control values. Clone miTMEM16F-10, furthermore, showed a reduced TMEM16K level as well (Fig. 1b). Whole cell patch clamp recordings of TMEM16A-5 showed a significant knockdown in current further supporting a successful knockdown of TMEM16A (Fig. 1c). This was not possible to do for TMEM16F, as the TMEM16A current was too dominating in ELA cells.
The role of TMEM16A and TMEM16F in cell migration
Analysis of cell migration in TMEM16A, 16F, and 16K KD clones
83.0 ± 5.8
88.9 ± 4.5
89.7 ± 6.9
81.2 ± 5.4
66.6 ± 4.8 **
48.5 ± 2.7***
51.0 ± 3.0***
53.2 ± 3.2***
54.7 ± 1.2***
Migratory speed (μm/min)
0.48 ± 0.03
0.53 ± 0.03
0.61 ± 0.03
0.65 ± 0.03**
0.44 ± 0.02**
0.30 ± 0.01***
0.29 ± 0.01***
0.32 ± 0.01***
0.29 ± 0.02***
Directional migration (μm)
66.7 ± 5.2
72.1 ± 4.2
72.4 ± 6.7
64.4 ± 5.8
54.3 ± 4.5**
36.4 ± 2.6***
40.3 ± 3.1***
34.8 ± 3.3***
43.5 ± 3.2***
0.72 ± 0.03
0.69 ± 0.02
0.59 ± 0.03*
0.53 ± 0.03*
0.62 ± 0.03*
0.68 ± 0.03
0.71 ± 0.02
0.68 ± 0.02
0.76 ± 0.02*
TMEM16A and TMEM16F impact on cell morphology
In the present study, we performed a detailed analysis of the contribution of different TMEM16 channel isoforms to migration of the mouse mammary ELA cell line. We focused on TMEM16A and TMEM16F channels that have the highest expression of TMEM16 channels in ELA cells. Due to the lack of specific pharmacological modulators, we employed a miRNA approach and stably knocked down the expression of TMEM16A and TMEM16F channels, respectively. The key result of our study is that TMEM16A and TMEM16F channels are both required for optimal cell migration. However, they appear to affect different components of the cellular migration machinery, so that their knockdown elicits different migratory phenotypes. TMEM16A channels are involved in the steering process, while TMEM16F channels are part of the migratory “engine”. Accordingly, knockdown of the two channels leads to impaired directionality and reduced translocation, respectively. The important question remains, by which mechanisms TMEM16A and TMEM16F elicit their differential effect on cell migration.
TMEM16A channels were shown to form Ca 2+-activated Cl− channels [4, 6, 29, 40] that can be activated by cell swelling . They can thereby act in concert with Ca2+ sensitive K+ channels (e.g., KCa3.1) in mediating localized cell shrinkage at the rear part of migrating cells [19, 32]. Since KCa3.1 channels were shown to be involved in directional migration , it is conceivable that TMEM16A knockdown impairs directionality too. In addition, TMEM16A channels could also modify the intracellular Ca2+ concentration by regulating the cell membrane potential. When TMEM16A channels are activated by a rise of the intracellular Ca2+ concentration, the resulting Cl− efflux will attenuate the hyperpolarization induced by the parallel activation of KCa3.1 channels. This would dampen Ca2+ influx by reducing the electrochemical driving force for Ca2+ ions. Along these lines, TMEM16A channels would affect the intracellular Ca2+ concentration in a similar way as TRPM4 channels . This implies that the intracellular Ca2+ concentration of miTMEM16A cells is higher than that of wt or mock-transfected cells. The small and roundish morphology of miTMEM16A cells could then at least in part be due to increased myosin contraction . In addition, the round morphology of miTMEM16A cells can also be explained on the basis of reduced Cl− efflux that is required for cell spreading .
TMEM16F channels are functionally quite distinct from TMEM16A channels. There is controversy about their biophysical properties. They are reported to constitute either a Ca2+-activated anion channel [19, 35] or a Ca2+-activated cation channel , and recently, Grubb et al. reported that PNa = 0.334(±0.028) PCl in TMEM16F overexpressing HEK cells . However, they also have nonconductive properties and mediate Ca2+-dependent phospholipid scrambling [37, 39] and thereby regulate the phospholipid distribution in the plasma membrane. Normally, phosphatidylethanolamine or phosphatidylserine are predominantly found in the inner leaflet of the plasma membrane. The asymmetric phospholipid distribution can be abrogated by the activity of Ca2+-dependent phospholipid scramblases [8, 37] or flippases . In the context of the present study, it is important to note that migration involves the translocation of phosphatidylethanolamine within the plasma membrane, since inhibition of the scramblases or flippases impairs migration [8, 16]. Along these lines, the inhibition of migration of miTMEM16F cells could also be a consequence of impaired phospholipid translocation.
The technical assistance of Sabine Mally, Birthe Juul Hansen, and Betina Larsen are gratefully acknowledged. This work was supported by the Marie Curie Initial Training Network IonTraC (grant agreement no. 289648) and by The Danish Council for Independent Research/Natural Sciences (grants 09–064182 and 10–085373) and Lundbeck Foundation, Denmark (J Nr R32-A3102).
Conflicts of interest
The authors declare that they have no conflict of interest.
All experiments were carried out in compliance to the current laws of the country.
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