Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20

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Recommended by Pavel Hozak Changes in intracellular calcium are necessary for the successful progression of mitosis in many cells. Both elevation and reduction in intracellular calcium can disrupt mitosis by mechanisms that remain ill defined. In
  Hindawi Publishing CorporationInternational Journal of Cell Biology Volume 2009, Article ID 487959, 12 pagesdoi:10.1155/2009/487959 Research Article CellCycle-DependentLocalizationof  Voltage-DependentCalciumChannelsandthe Mitotic Apparatus ina NeuroendocrineCellLine(AtT-20) KarenJ. Loechner, 1  Wendy C.Salmon, 2 Jie Fu, 3 ShipraPatel, 1 andJames T. McLaughlin 3 1 Division of Pediatric Endocrinology, Department of Pediatrics, University of North Carolina Chapel Hill, Chapel Hill, NC 27599, USA  2  Michael Hooker Microscopy Facility, University of North Carolina Chapel Hill, Chapel Hill, NC 27599, USA 3 Department of Pharmacology, University of North Carolina Chapel Hill, Chapel Hill, NC 27599, USA Correspondence should be addressed to Karen J. Loechner, karen loechner@med.unc.eduReceived 4 March 2009; Revised 13 July 2009; Accepted 10 October 2009Recommended by Pavel Hozak Changes in intracellular calcium are necessary for the successful progression of mitosis in many cells. Both elevation andreduction in intracellular calcium can disrupt mitosis by mechanisms that remain ill defined. In this study we explore the role of transmembrane voltage-gated calcium channels (CaV channels) as regulators of mitosis in the mouse corticotroph cell line (AtT-20). We report that the nifedipine-sensitive isoform CaV1.2 is localized to the “poleward side” of kinetechores during metaphaseand at the midbody during cytokinesis. A second nifedipine-sensitive isoform, CaV1.3, is present at the mid-spindle zone intelophase, but is also seen at the midbody. Nifedipine reduces the rate of cell proliferation, and, utilizing time-lapse microscopy,we show that this is due to a block at the prometaphase stage of the cell cycle. Using Fluo-4 we detect calcium fluxes at sitescorresponding to the mid-spindle zone and the midbody region. Another calcium dye, Fura PE3/AM, causes an inhibition of mitosis prior to anaphase that we attribute to a chelation of intracellular calcium. Our results demonstrate a novel, isoform-specific localization of CaV1 channels during cell division and suggest a possible role for these channels in the calcium-dependentevents underlying mitotic progression in pituitary corticotrophs.Copyright © 2009 Karen J. Loechner et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited. 1.Introduction Voltage-dependent calcium channels (CaV channels) aremultisubunit transmembrane proteins that are mediators of entry of extracellular calcium ions into cells of nerve, muscle,and endocrine tissues [1]. Genomic studies have identified3 families for the ten genes that encode the alpha1 subunitsdesignated CaV1, CaV2, and CaV3 [2]. The diversity of CaVchannel genes allows for a large number of channel isoforms,and these di ff  erent isoforms are often expressed in the samecell. By mediating changes in intracellular free calcium,CaV channels act as key mediators of signaling events suchas cell depolarization, neurotransmitter and neuropeptidesecretion, and regulation of gene expression [3, 4]. An important objective in calcium channel biology, therefore, isto understand the specific role(s) for each channel isoform,and their integration in di ff  erent cellular events.One well-established role of calcium channels is the cou-pling of membrane depolarization to release of neurotrans-mitters [5, 6]. Both CaV2.1 and CaV2.2 have been shown to interact directly with, and be modulated by, proteinsthat comprise the neurotransmitter release apparatus (theSNARE complex). Colocalization of channels and the releasemachinery facilitates coupling between the active calciumchannels and the calcium dependent fusion of transmitter-containing vesicles with plasma membrane.In neuroendocrine cells, a similar coupling betweenCaV1 channels and release machinery is thought to underliesecretion of peptides such as insulin, growth hormone, orACTH [7, 8]. The pituitary corticotroph cell line, AtT-20, is a well-establishedmodelsystemforstudiesofACTHsecretion.These cells express multiple isoforms of CaV, yet only theCaV1 channels are coupled to CRH- or depolarization-stimulated secretion of ACTH [9, 10]. In a recent study we  2 International Journal of Cell Biology examined the cellular distribution of CaV1 channels andSNARE proteins in AtT-20s cells and found colocalization of CaV1.2, but not CaV1.3, with components of the synapticmachinery and releasable peptide [11]. In the course of thisstudyweobservedCaV1channelslocalizednearcomponentsof the mitotic apparatus in dividing cells. These observationssuggested that the AtT-20 cell could provide a useful modelto examine the possible role for CaV1 channels in anothercellular function, mitosis.A role for calcium signaling in mitosis has been inferredfor decades, yet the mechanism underlying calcium elevationduring cell division has, to date, not been elucidated. Studieshave established a role for calcium and/or demonstratedalterations in calcium gradients during mitosis [12–19]. Calcium is involved in regulating mitotic checkpoints; thecritical point at which progression through mitotic stages isclosely regulated has also been shown [20–24]. Furthermore, the role of calcium-dependent kinases in mitosis has alsobeen examined (reviewed in [25]).The CaV channels observed in dividing AtT-20 cellsrepresent a possible contributor to intracellular calciumfluxes during mitosis. Antagonists to the CaV1 subtype(dihydropyridines (DHPs)) have been reported to block mitosis in a number of systems [26–29], as so it is possible that these channels play a role in the mitotic process.While the limited access to the mitotic apparatus couldpresent a barrier to some drugs, DHPs are highly lipophilic[30] and therefore could reach internal membrane sites. Inthis paper, we show that CaV1 channels are localized inclose proximity to mitotic structures. We then use severalexperimentalapproachestoassesswhetherthesechannelsareactive participants in cell division in AtT-20 cells. 2.Methods  2.1. Cell Culture.  AtT-20/D16v cells are from American TypeCulture Collection (ATCC, Manassas, VA). All cell culturereagents are obtained from Invitrogen (Carlsbad, CA). AtT-20 cells are cultured in Dulbecco’s Modified Eagle medium(DMEM) containing 4.5gm glucose/L and 10 percent fetalbovine serum. Cells are maintained at 37 ◦ C in a 5% CO2humidified incubator and passaged as described previously [9].Formicroscopy,cellsareplatedonchamberedcoverslips(Labtek/Nunc, Rochester NY) and grown 2 to 4 days priorto fixation. For proliferation assays, cells are plated in 8-wellplates (for cell counting) and 96-well plates (for MTS assay)to be used 2–4 days after plating ( ∼ 60%–90% confluence).For calcium and time-lapse imaging, cells are plated 2–4days prior to experiments on coated glass bottom microwelldishes (Mattek Corp, MA). Cells are then transferred tosteroid-free media (Charcoal/Dextran treated FBS; Hyclone,Logan UT) 12–18 hours prior to fixation.  2.2. Immunocytochemistry.  Cells are rinsed briefly in Phosphate-Bu ff  ered Saline (PBS), and then fixedby incubation 20 minutes in PBS containing 4%paraformaldehyde. Following three washes with PBS,cells are incubated 5 minutes in 100% methanol at 20 ◦ C.They are washed again in PBS, and then permeabilized inPBS containing 20mM NaN3, 0.2% Tween-20, and 0.5%NP-40. All subsequent washes employ Antibody WashBu ff  er (WB; PBS plus 0.2% Tween-20 and 0.05mg/mLBovine serum albumin (BSA, IgG free; Sigma, St. LouisMO). Fixed cells are incubated 24–48 hours at 4 ◦ C withone or more primary antibodies diluted into WB (antibody sources listed below). Following 5 washes with WB, cells areincubated with the appropriate combination of fluorescentsecondary antibodies (Alexa-fluor conjugates, Invitrogen,Carlsbad CA) for 1 hour at 22 ◦ C. This is followed by 3washes, and a final rinse with PBS. Stained cells are stored at4 ◦ C in Slo-fade (Invitrogen); or in Slo-fade containing thenuclear counterstain DAPI (4  -6-Diamidino-2phenylindole;Invitrogen). Primary antibodies are obtained from thefollowing commercial sources: Antibodies to CaV1 channelalpha1 subunits are from Alomone Labs (Jerusalem, Israel);Anticentromere (CREST) antibody was from AntibodiesIncorporated (Davis, CA); anti- α  tubulin, clone DM1Afrom Sigma (St. Louis, MO). The antibodies to CaV1channel alpha1 subunits are raised in rabbits against peptidesequences specific to the CaV1.2 (peptide sequence: TTKINMDDLQ PSEN EDKS) and CaV1.3 (peptide sequence:DNKVT IDDYQ EEAE DKD) isoforms. A basic localalignment search (Blast; National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/) of all non-redundant protein sequence databases shows no knownprotein other than these with greater than 50% sequencesimilarity to either of these sequences; similar searches of the mouse protein database identify the peptide sequencesonly in these two channel polypeptides. In tests of specificity,we detect no specific CaV1 staining when the primary antibody is omitted from the protocol, or when the antibody is preadsorbed to a ∼ 10-fold molar excess of peptide antigen(not shown).  2.3. MTS Assay.  Cells are plated at low density in 96-wellplates (5 × 10/well), allowed to grow for 24–48 hours, andthen incubated 72 hours in media to which nifedipine isadded at the concentrations indicated. All wells contain thesame concentration of vehicle, 0.05% DMSO. Cell density is measured using a modified methyl tetrazolium protocol(MTS; Promega Madison WI) according to manufacturer’sinstructions. Data is normalized to absorbance of cellsincubated with vehicle alone and plotted as a function of nifedipine concentration. All graphs are plotted using Originv8 (OriginLab, Northampton MA).  2.4. Confocal Microscopy.  We image stained cells at multiplewavelengths using a Zeiss 510 Meta laser scanning confocalwith inverted microscope stand (Carl Zeiss, Jena, Germany)using a 63X 1.4NA oil immersion objective. Mitotic stagesare detected by eye using DAPI to visualize chromosomemorphology; due to the limitation of the confocal systemto 3 color channels, imaging of chromosomes was notpossible in addition to the proteins described. All channelsare imaged sequentially, and images are stored as chunky RGB files. Figures are assembled using Adobe Photoshop  International Journal of Cell Biology 3(ver. 7.0). Images with four labels (Figure 1(A)) display some bleedthrough of the microtubule staining into theCRESTstaining.WecorrectedforthisusingMetaMorphv4.5software (Molecular Devices, Sunnyvale CA) by calculatingthe relative intensity of microtubules in both the CRESTand microtubule images, multiplying the microtubule imageby the value and subtracting the resultant image from theCREST image.  2.5. Visual Mitotic Stage Assay.  Cells are plated at low density (5  ×  10 3 /well) in chambered cover slips (Labtek/Nunc,Rochester NY), allowed to attach for 24–48 hours, and thenincubated either 24 or 72 hours in media containing Nifedip-ine (6 µ M). Multiple fields (minimum of 5 per condition) of AtT-20 cells at similar cell density are randomly selected andimaged with a Hamamatsu ORCA-ER camera mounted on aLeica DMIRB inverted microscope equipped with a mercury lamp and DAPI filter cube and 40X 1.25NA objective. Thenumber of cells in each stage is determined using “Manually Count Objects” application in MetaMorph. We group cellsidentified in anaphase, telophase, or cytokinesis into a singleclass (A/T/C) in our analysis due to the low mitotic index and the di ffi culty in di ff  erentiating among these phases usingchromosome morphology alone.  2.6. Time-Lapse Microscopy.  Time series of DIC imagesare acquired as described above using a 30-second timeinterval. Cells are maintained in covered dishes at 37 ◦ C overthe course of the imaging session to minimize changes inosmolarity. We define a significant mitotic “block” as any stage in which time elapsed is equal to or greater thantwo standard deviations of the mean time required for thatstage by control cells. For Fura PE3/AM AtT-20 cells areimaged by DIC on a Nikon TE2000U inverted microscopewith a 100X 0.5–1.3NA S Fluor objective with the iriscollar set to 1.3NA using a DVC 1312 monochrome CCDcamera (DVC Co.) controlled with Simple PCI software(v5.3, Hamamtsu, Inc.). Cells are kept at 37 ◦ C using an aircurrent incubator (AirTherm). To ascertain that the e ff  ectson cell cycle progression are not a function of the imagingsetup or cell culture conditions, a prophase cell is imaged forthe entire mitotic cycle ( ∼ 1.5 hours) without the additionof Fura PE3/AM (4 µ M) on each experimental day. We thenexclude all data collected on days when the control cellsdo not complete the entire mitotic cycle within the meantime  ± 1 S.D. Experimental dishes are incubated with FuraPE3/AM(4 µ M)at37 ◦ Cfor20minutespriortoDICimagingof mitosis versus vehicle controls (DMSO).  2.7. Calcium Imaging.  Fluo-4 fluorescence is imaged in livecells on a Nikon TE2000U inverted microscope a DVC1412 Intensicam II. The DIC and fluorescence images areacquired simultaneously using a long pass 610nm filter forthe DIC illumination, a dual camera adapter connectedto the camera port, and a 565nm dichromatic mirror inthe dual-camera adapter to direct the fluorescence emissionlight to the DVC 1412 Intensicam II and the DIC imageto a DVC 1312 camera. A Sutter DG-4 xenon arc lampsource and fast filter changer are used for illumination of fluorescence and selection of excitation wavelengths. AnFITCfiltersetismountedinthemicroscopestandwithwhitelight delivered from the DG-4. Fluorescence illuminationandallimageacquisitionsarecontrolledusingtheSimplePCIimage acquisition software running on two PC computers,one for each camera. To minimize phototoxicity to the cellsfrom excessive fluorescence excitation, every one minutea set of 10 images is acquired at a 1 second interval.The rapidity of the acquisition pattern is due to the shortduration of anaphase (minutes). Signal intensity of multipleareas in the time-lapse image series is determined usingthe intensity measurement functions in the SimplePCIsoftware. 3.Results 3.1. CaV1.2 and CaV1.3 Localize to the Mitotic Appara-tus.  Figures 1(A) and 1(B) show the pattern of staining observed for CaV1.2 and CaV1.3, respectively, for AtT-20cells captured and fixed at di ff  erent stages in mitosis. Tovisualizethekinetechoresinmetaphase,cellsarestainedwitha kinetechore antibody (CREST antibody) in addition toantibodies to CaV1.2 and CaV1.3. Costaining demonstratesthat CaV1.2 localizes to sites adjacent to the “poleward” of the kinetechores in both metaphase and anaphase (Figures1(A), (a), (b), inserts). Of note, this distribution of CaV1.2with kinetechores during metaphase is seen throughout thevolume of the cell as imaged using z-stacking across theentirety of the spindle apparatus (not shown) and supportsthat the localization is within the AtT-20 cell and notsuperimposed imaging from surface proteins.To visualize the microtubules of the mitotic spindle, cellsare also costained with the  α -tubulin antibody DM1A. Intelophase, we observed CaV1.3 (Figure 1(B), panel (a)) atthe mid-spindle zone of the mitotic apparatus; we do notobserve a similar pattern with CaV1.2 (Figure 1(A), panel(a)). However, as the microtubules condense further to formthe midbody during cytokinesis, both CaV1.2 and CaV1.3are then visualized at the midbody (Figure 1(A) panel (c),and Figure 1(B) panel (b)). These staining patterns suggestthat the cells direct an active, isoform-specific redistributionof CaV1 channels over the course of the cell division cycle.To test if the mitotic channel distribution occurs in othercell types, we examined the distribution of CaV1 channelsin two additional cell lines of neuroendocrine srcin: PC12pheochromocytoma cells and INS-1 beta pancreatic cells.Both of these cell types are known to express CaV1 channels[31, 32]. Figure 1 shows that, as in AtT-20 cells, both CaV1.2 (Figure 1(A) panel (d)) and CaV1.3 (Figure 1(B) panel (c)) are localized to the midbody in PC12s during cytokinesis. Incontrast, we did not see a similar pattern of CaV1 stainingin INS-1 cells undergoing mitosis; the CaV1.3 channelsare visible at midbody whereas the CaV1.2 channels arenot (Figure 1(A), panel (g); Figure 1(B), panel (e)). Specific staining for both isoforms was readily visible in interphasecellsasreportedpreviously[31].Thesedi ff  erencesinchannelisoform staining support the idea that the patterns weobserve are due to real di ff  erences among neuroendocrine  4 International Journal of Cell Biology  CaV1.2 Crest Microtubules Merge     A    t    T  -    2    0    M   e    t   a   p     h   a   s   e    A    t    T  -    2    0    A   n   a   p     h   a   s   e    A    t    T  -    2    0    C   y    t   o     k    i   n   e   s    i   s    P    C    1    2    C   y    t   o     k    i   n   e   s    i   s    I    N    S  -    1    M   e    t   a   p     h   a   s   e    I    N    S  -    1    A   n   a   p     h   a   s   e    I    N    S  -    1    C   y    t   o     k    i   n   e   s    i   s 5 µ m5 µ m5 µ m5 µ m5 µ m5 µ m5 µ m(a)(b)(c)(d)(e)(f)(g)R G B M     2       × R G B M     2       × (A) Figure  1: Continued.  International Journal of Cell Biology 5 CaV1.3 Microtubules Merge     A    t    T  -    2    0    T   e     l   o   p     h   a   s   e    A    t    T  -    2    0    C   y    t   o     k    i   n   e   s    i   s    P    C    1    2    C   y    t   o     k    i   n   e   s    i   s    I    N    S  -    1    T   e     l   o   p     h   a   s   e    I    N    S  -    1    C   y    t   o     k    i   n   e   s    i   s 5 µ m5 µ m5 µ m5 µ m5 µ m(a)(b)(c)(d)(e)(B) Figure  1: Continued.
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