| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
1Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington; and 2Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois
Submitted 9 October 2006 ; accepted in final form 29 January 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca2+-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca2+-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca2+-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca2+-ATPase. sarco(endo)plasmic reticulum calcium-adenosine triphosphatase; differentiation; C2C12 myocytes; vesicle trafficking
-adrenergic stimulation by means of the phosphorylation of PLB through either cAMP- or CaM-dependent kinases (3, 18, 26, 27, 31). PLB and the Ca2+-ATPase reside as an integral membrane protein complex in the SR, where maximal inhibition of the Ca2+-ATPase requires an equal stoichiometry of PLB, as is present in coexpressed or reconstituted vesicles, as well as in vivo in SR isolated from slow-twitch skeletal muscle (8, 20). However, in the heart, PLB is present in a three- to fourfold molar excess relative to the Ca2+-ATPase, suggesting that this reserve pool of free PLB provides a graded response of calcium transport rates to cellular kinases. The functional importance of maintaining optimal expression levels of PLB relative to that of the Ca2+-ATPase is highlighted by variations in PLB-to-Ca2+-ATPase ratios that correlate with defective calcium regulation in hyper- and hypothyroidism, aging, and the failing heart (16, 17). Furthermore, genetic manipulations have demonstrated that cellular changes in PLB-to-Ca2+-ATPase molar ratios can act to compensate for altered calcium affinities of Ca2+-ATPase isoforms (33). The initial formation of PLB-Ca2+-ATPase interactions in the developing heart is coincident with progressively decreasing stoichiometries of total expressed PLB relative to Ca2+-ATPase as differentiation to mature myocytes occurs (2, 24). PLB is expressed early in the undifferentiated myocyte, whereas upregulation of the Ca2+-ATPase occurs coincidentally with differentiation and the formation of the SR membrane, which is a specialized region of the endoplasmic reticulum (ER). In addition, differentiation requires new trafficking pathways for these proteins from their site of synthesis in the ER to the newly forming SR. However, little is known regarding the routing of these or any SR resident proteins from ER to SR, or how the crucial interactions between PLB and the Ca2+-ATPase are formed in either immature or adult myocytes.
Therefore, to understand the cellular mechanisms associated with targeting PLB to the SR in developing muscle, we investigated the coordinated expression of PLB during myocyte differentiation using the C2C12 muscle cell line, which is amenable to transfection with a variety of fusion proteins with fluorescent labels. By monitoring the localization and cellular trafficking of PLB and the Ca2+-ATPase, we found that both endogenous and transfected Ca2+-ATPase exhibit a reticular localization that is consistent with an ER/SR localization, whereas PLB is present as punctate vesicles in myoblasts that undergo directed trafficking to the plasma membrane before their degradation. Following differentiation, PLB colocalizes with the Ca2+-ATPase in the SR of myotubes. These results indicate an important role for cellular trafficking in mediating the formation of a functional SR associated with cellular signaling and suggest a possible role for PLB in the plasma membrane associated with muscle development.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Antibodies to SERCA2a, SERCA1, and PLB were obtained from Affinity BioReagents (Golden, CO), and antibodies to golgin-97 and Alexa Fluor 594 goat anti-mouse IgG were obtained from Invitrogen (Carlsbad, CA). The H2 kinesin heavy chain antibody has been described previously (25). The monoclonal antibody (KLC-2B) recognizing a subset of kinesin light chain-1 alternatively spliced isoforms (KLC-B and KLC-C; Ref. 6) was generated by immunizing mice with a peptide (MRKMKLGLVK) found exclusively in these kinesin light chain-1 isoforms and producing antibodies as described previously (25). These antibodies have been extensively characterized and recognize a subset of native kinesin light chains and bacterially expressed kinesin light chains B and C, but not A (28). The MHC monoclonal antibody (MF-20) developed by Donald A. Fischman was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa (Department of Biological Sciences, Iowa City, IA 52242). Mitotracker red dye was obtained from Invitrogen, and mitochondria in live cells were labeled according to the manufacturer's instructions. BG-505, BG-DAF, and BG-TMR were obtained from Open Biosystems (Huntsville, AL). All cell culture media, unless otherwise stated, were obtained from GIBCO Laboratories (Grand Island, NY).
AGT-tagged proteins. O6-alkylguanine-DNA alkyltransferase (AGT)-tagged proteins were expressed in myocytes from AGT utility vectors created using PCR to amplify the AGT coding sequence from the pSS26m vector (Covalys, Witterswil, Switzerland) with primers containing 5' Age1 and NotI sites and a 3' BsrG1 site. The enhanced GFP (EGFP) coding sequence was replaced with the PCR-amplified AGT sequence by using the Age1 and BsrG1 restriction sites to create the vector pAGT-C1 containing the same multicloning site as the original pEGFP-C1 vector. PLB and SERCA were directly subcloned from the corresponding pEGFP-based vectors into the pAGT-based vectors. Retroviral vectors containing AGT-PLB and AGT-SERCA were created by inserting the Not1/BamH1 digest into the pBM retroviral vector (originally from the Garry Nolan Lab, Stanford University, Palo Alto, CA) modified by expansion and inversion of the multiple cloning site. C2C12 stable cell lines expressing tagged PLB and SERCA1a were generated by selecting transduced cells in puromycin.
Tetracysteine-tagged PLB. The plasmid pTC-C1 was made by synthesizing two oligonucleotides containing 5' AgeI and NotI sites and a 3' BsrG1 site, separated by a DNA sequence encoding the amino acids MAEAAAREACCPGCCARAR. The two oligos were annealed and digested with AgeI and BsrG1, and this fragment was inserted into the Age1 and BsrG1 sites in pEGFP-C1. The PLB sequence was inserted as described above for the AGT vector. As described for AGT-tagged PLB, a retroviral vector coding for this tetracysteine (TC)-tagged PLB was created and stably expressed in C2C12 cell lines.
Cell culture. C2C12 skeletal myocytes (CRL-1722; ATCC, Rockville, MD) were grown at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS; GIBCO), penicillin/streptomycin, 1 mM sodium pyruvate, and 4 mM L-glutamine. For differentiation experiments, this growth medium was replaced with differentiation medium consisting of DMEM, 2% horse serum, penicillin/streptomycin, 1 mM sodium pyruvate, and 4 mM L-glutamine after the cells reached confluency. Additional myocyte cell lines tested (Sol8, L6, and H2C9) were obtained from ATCC and grown under identical conditions.
Immunoblotting and immunofluorescence. Cell lysates were prepared using 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (wt/vol) Nonidet P-40, 5% glycerol, 10 µM leupeptin, 2 µg/ml aprotinin, 1 µM pepstatin, 2 mM PMSF, 50 mM NaF, and 1 mM Na3VO4. These lysed cells were centrifuged (30 min at 16,000 g), and the protein concentration in the lysates was determined before electrophoretic separation. SDS-PAGE was performed using 7.5, 15, or 4–12% polyacrylamide gels. Proteins were transferred to a polyvinylidene difluoride membrane using a semidry transfer cell (Bio-Rad) for 30 min at 25 V for SERCA1 and SERCA2 identification and for 15 min at 20 V for PLB identification, in buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol (for SERCA) or 10% methanol (for PLB). For detection of SERCA2a, blots were probed with anti-SERCA2a monoclonal antibody 2A7-A1, which recognizes residues 386–396 of canine SERCA2a (12), with dilution 1:10,000. SERCA1 isoform was detected using anti-SERCA1 monoclonal antibody IIH11 with dilution 1:20,000. PLB was detected with anti-PLB monoclonal antibody 2D12 with dilution 1:2,000. Secondary antibodies conjugated with horseradish peroxidase were used, and proteins were detected using enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ). To detect endogenous proteins in myocytes, we carried out immunolabeling as previously described (29) using the above antibodies. Primary antibodies were detected using Alexa Fluor 594 goat anti-mouse IgG from Invitrogen.
Live cell imaging and fluorescence recovery after photobleaching. Live cell microscopy was performed on cells transiently transfected with GFP-, CFP-, or YFP-tagged PLB or SERCA1a as indicated. Cells were either fixed for colabeling with antibodies or imaged live using a Nikon TE2000 inverted microscope equipped with stage and lens heaters (Bioptechs, Butler, PA). To label AGT-tagged proteins, we incubated cells in medium containing 4 µM benzyl guanine (BG)-505, BG-diacetylfluorescein (DAF), or BG-tetramethylrhodamine (BG-TMR) star for 30 min, followed by a 15-min wash in medium before live cell analysis or fixation. For dual-color pulse-chase experiments, cells were labeled with BG-TMR, washed, and incubated for 5 h in ligand-free cell medium before being relabeled with BG-505. BG-505 was visualized using fluorescein filter sets, whereas BG-TMR was imaged using rhodamine filter sets. Labeling TC-PLB-expressing cells involved incubation of cells for 30 min in HEPES-buffered saline (HBS) containing 1 µM FlAsH·EDT2 reagent. Cells were washed with HBS, incubated for 5 min in HBS containing 5 mM EDT to reduce nonspecific background, and washed twice in HBS before replacement of tissue culture medium and imaging of live cells. The supplemental movie showing movement of AGT-PLB was acquired using Metamorph software (Universal Imaging, Downington, PA) to acquire a stack of time-lapse images and saved in Quicktime format. (The online version of this article contains supplemental data.)
Fluorescence recovery after photobleaching (FRAP) was performed using a Leica confocal microscope equipped with an argon laser and appropriate filters for imaging GFP-tagged proteins. For photobleaching, a rectangular region of the desired size was photobleached using 100% laser power for a total duration of 
1 s. Images were collected before, immediately after, and at set intervals following the bleach for a time sufficient to allow complete recovery of each protein. Original images were exported as tagged image files, and final illustrations were assembled using Adobe Photoshop.
Red fluorescent protein endosomal marker. As a marker of endosomes, the RFP-Endo vector was created by inserting the RhoB GTPase (from pEYFP-Endo; Clontech) into a pBM retroviral vector containing a red fluorescent protein (RFP) tag using PCR. A double stable cell line expressing AGT-tagged PLB and RFP-Endo was created using two retroviral vectors containing different selection markers (puromycin for AGT-PLB and blasticidin for RFP-Endo).
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
90% confluency in growth medium (DMEM with 10% FBS) before its replacement with differentiation medium (DMEM with 2% horse serum) for 1–7 days. As shown in Fig. 1B, differentiation had no significant effect on total PLB expression levels, but both SERCA1a and SERCA2 showed a linear increase in abundance as cells differentiated. This increase in SERCA abundance coincided with increased proteins levels of myosin heavy chain (MHC), a classic marker of muscle differentiation (data not shown). We note that this differential expression pattern was also observed in other myocyte cell lines tested, i.e., the L6 and Sol8 skeletal myocytes and the cardiac H2C9 cell line (data not shown), indicating that the early appearance of PLB in myoblasts in the absence of the Ca2+-ATPase is a general feature of cultured myocytes, and this pattern mirrors the same changes in relative abundances of PLB and the Ca2+-ATPase observed in the developing heart (2, 24).
|
Localization of GFP-PLB and GFP-SERCA1a. To better address intracellular trafficking of PLB and SERCA in living myocytes, we transiently transfected C2C12 myoblasts with GFP-PLB or GFP-SERCA1a, essentially as described by Biehn et al. (4). The distribution of GFP-PLB (Fig. 2, top left), like that of endogenous PLB (Fig. 1), exhibited a punctate pattern that extended to the edge of the cell and localized to regions of the plasma membrane and cellular extensions in many cells. On the other hand, in myoblasts, GFP-SERCA1a (Fig. 2, bottom left) was constrained within, but evenly distributed throughout, the endoplasm and was observed to be reticular in many regions. This reticular intracellular distribution is similar to that expected for wild-type SERCA in differentiated muscle, i.e., in the SR, and mirrors that observed when exogenous GFP-SERCA1a is expressed in fibroblasts, demonstrating that in the absence of an SR membrane, SERCA1 is targeted to the ER (4).
|
To further compare the intracellular trafficking and turnover of PLB and SERCA, we created stable cell lines expressing PLB and SERCA, each as fusion proteins with AGT. This genetically engineered version of AGT is a 21-kDa protein that cleaves cell-permeable para-substituted benzyl guanines (BG) to create a stable thioether intermediate that provides a fluorescent group covalently bound to PLB or SERCA and is suitable for live cell imaging (14, 15). These stable myocyte cell lines were amenable to differentiation into myotubes in which the levels of expressed AGT-PLB and AGT-SERCA protein were 50% of that of endogenous (wild type) PLB and SERCA (data not shown). As shown in Fig. 2, middle, C2C12 myoblasts expressing AGT-PLB and labeled with a BG fluorescein derivative, BG-505, showed the same punctate distribution as endogenous or GFP-PLB, which is distinct from the reticular distribution of SERCA. Finally, as an additional control for the possibility that the large size of either the GFP (30 kDa) or AGT (21 kDa) tags relative to the smaller PLB (6 kDa) might produce artifactual aggregation, a PLB construct was genetically engineered with an NH2-terminal sequence containing a small TC motif suitable for labeling with the cell-permeable FlAsH (<700 Da; Ref. 9). This TC-tagged PLB (C4-PLB) expressed in C2C12 myoblasts and labeled with FlAsH also exhibited a punctate fluorescence pattern (Fig. 2, right). Thus the punctate distribution of PLB in undifferentiated myoblasts consistently observed for wild-type PLB in fixed and immunostained cells as well as PLB expressed with both large and small tags for live cell imaging provides a strong level of confidence that PLB localization in myoblasts is distinct from an ER localization or its reticular localization in the mature myotube.
Differential mobilities of PLB and SERCA in myoblasts.
As an alternative to cell imaging, we addressed the differential localization of PLB and SERCA by biophysical measurements of FRAP, since rates of lateral protein mobility can be altered by cell locale. As shown in Fig. 3A, when a region of a myoblast expressing GFP-SERCA1a was photobleached, a gradual, uniform recovery of fluorescence was observed. The half-life of recovery was 20 s, significantly slower than the half-life of freely diffusing GFP (0.3 s; Ref. 30). Thus the mobility of SERCA1a is constrained, consistent with its lateral diffusion within the ER membrane. In contrast, when FRAP was performed on GFP-PLB, a much different recovery pattern was observed (Fig. 3B). First, the recovery occurred more slowly, requiring several minutes for complete recovery of the fluorescence. Second, the GFP-PLB entered the bleached area as discrete, subcellular structures, which suggests recovery is due to transport of GFP-PLB-containing vesicles into the bleached zone (see magnified images in Fig. 3C). Thus these very different rates and modes of diffusion of SERCA compared with PLB are incompatible with colocalization, indicating their localization to distinct compartments, i.e., ER and vesicles, respectively, in undifferentiated myocytes.
|
Differential turnover of PLB and SERCA. As a means to monitor the localization of PLB and SERCA over time after protein synthesis, we took advantage of the covalent labeling of AGT-tagged proteins with BG-derivatized fluorophores, which provides a means to pulse label a population of proteins. After labeling, changes in the location and intensity of the fluorescence signal associated with either PLB or SERCA are attributable to either trafficking or protein turnover and are insensitive to the biosynthesis of new proteins. To determine the relative turnover rates of PLB and SERCA1a, AGT-PLB- or AGT-SERCA-expressing myoblasts were labeled with a pulse of BG-fluorescein, which labels all expressed protein. After a wash step to remove unbound fluorophore, both the levels of labeled protein and their cellular distribution were monitored over time (Fig. 4A). As illustrated in Fig. 4A, top, the fluorescence associated with labeled PLB disappeared within 24 h and was accompanied by a noticeable time-dependent change in the distribution of the labeled PLB within the cell. Specifically, fluorescence at the plasma membrane disappeared relatively early on, and over time the fluorescence became less intense but more centrally localized around the nucleus. In contrast, fluorescently labeled AGT-SERCA exhibited a relatively stable reticular distribution over the time course of the experiment given that its fluorescence intensity diminished, consistent with the synthesis and retention of AGT-SERCA as an integral ER membrane protein. Moreover, fluorescence associated with SERCA persisted for over 24 h (Fig. 4A), consistent with the 30-h half-life for SERCA1 previously measured with radioisotopes in chick myocytes (11). The more rapid loss of fluorescence associated with AGT-PLB compared with that of AGT-SERCA suggests its shorter protein half-life than that of SERCA; to our knowledge, protein turnover of PLB has not been previously measured.
|
Altered PLB distribution occurs upon myocyte differentiation. The increased expression of SERCA in differentiating myocytes or developing muscle correlates with proliferation of newly formed SR. Therefore, the change in PLB localization as myocytes differentiate could be due to recruitment of PLB to SERCA as it becomes available in the SR. To test whether the expression of SERCA in myoblasts affects PLB localization, we cotransfected myoblasts with bioluminescent forms of PLB and either SERCA1a or SERCA2a and examined their intracellular distribution in individual cells. As shown in Fig. 5A, YFP-PLB retained its vesicular distribution even in the presence of coexpressed CFP-SERCA1a. Coexpression of SERCA2a, which directly interacts with PLB in vivo, also did not affect the localization of PLB in myoblasts. In this case, untagged SERCA2a was utilized to avoid any potential interference of a tag with PLB-SERCA2a interactions (Fig. 5B). Thus the presence of SERCA2a or SERCA1a in the ER of the myoblast is not sufficient to stimulate the redistribution of PLB into the reticular compartment and to co-localize with SERCA.
|
Colocalization of AGT-PLB with cellular reference markers in live cells. Since PLB does not show substantial localization in the ER in undifferentiated cells, we performed colocalization experiments with cell compartment-specific dyes and antibodies to identify the cellular compartment(s) associated with PLB trafficking. As shown in Fig. 6A, a concentration of AGT-PLB was observed in the region of the Golgi, as evidenced by colocalization of a subset of PLB in the perinuclear region stained with an antibody to the Golgi resident protein golgin. In contrast, there was no significant colocalization of PLB with mitochondria when this compartment was labeled with Mitotracker red (data not shown), indicating that PLB is excluded from mitochondrial membranes. Additional resolution regarding the possible role of endosomes in mediating PLB trafficking was addressed through an assessment of the possible colocalization of AGT-PLB (shown labeled with BG-505 in green) and an RFP-endosome marker, i.e., the RhoB GTPase (Fig. 6, B and C). We found extensive colocalization of PLB with the labeled endosomes, as highlighted in the enlarged image that shows many vesicles containing both AGT-PLB and the RFP-Endo (Fig. 6C).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Of note, although a large fraction of PLB is associated with vesicles, it is apparent from the low level of background fluorescence associated with both endogenous and tagged PLB in our images that a fraction of PLB is associated with ER (Figs. 1–6). This observation is consistent with the expression of SERCA2b in myoblasts and with prior observations regarding the ability of PLB to regulate SERCA2b when coexpressed in cells and in genetically altered mice (34, 35). Thus PLB may have a dual role in the myoblast, one to regulate SERCA2b in the ER, and a second, as yet unidentified, that involves extensive trafficking of PLB to the plasma membrane.
One surprising result of this study is that it is not the availability of SERCA2a that is responsible for the localization of PLB to the ER as evidenced by the observation that the distribution of PLB in vesicles was retained in the presence of exogenous SERCA2a expressed in myoblasts at levels comparable to those of differentiated myotubes. Only upon the extensive reorganization associated with myocyte differentiation and the formation of SR membranes did the bulk of PLB colocalize with the Ca2+-ATPase, suggesting an important role for intracellular signaling processes in mediating the development of functional SR membranes that contain optimal amounts of PLB and the Ca2+-ATPase. Although the possibility that the same myoblast trafficking pathway of PLB-containing vesicles also occurs in differentiated myotubes has not been explicitly addressed in the present study, the inability to detect PLB-containing vesicles in myotubes implies that PLB, like the Ca2+-ATPase, resides primarily in the SR after differentiation. However, the possibility cannot be ruled out that the extensive proliferation of SR in myotubes masks ready detection of a minor vesicular population of PLB that trafficks from ER to plasma membrane (Fig. 7).
|
Differentiation requires that both PLB and SERCA transit to the SR from their site of synthesis in the ER. The associated mechanisms for these or other SR resident proteins are not well understood, but current work has suggested that this relocation does not involve direct lateral diffusion through the membrane bilayer as previously thought. For example, the SR lumenal protein calsequestrin has been shown to be routed to the SR via COPII vesicles budding at ER exit sites, but without Golgi involvement (23). Similarly, SERCA in the heart has been suggested to be routed to SR via vesicles by an ankyrin-B-assisted protein sorting mechanism (32). The route that PLB takes has not yet been identified, but the excess stoichiometry of PLB to SERCA in the heart and its faster protein turnover rate suggests that PLB is transported independently from SERCA.
In summary, a combination of new and established imaging tools has revealed the developmentally regulated trafficking of PLB in the myoblast that is distinct from that associated with the Ca2+-ATPase, its interaction partner in the mature myocyte. These observations suggest possible new roles for PLB in the myoblast and highlight the important cellular regulation associated with the trafficking of PLB in the formation of fully functional SR membranes in which the Ca2+-ATPase is under -adrenergic control through its association with PLB. Future measurements should focus on understanding the relationship between the trafficking of PLB and the maturation and maintenance of cardiac function.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Arai M, Otsu K, MacLennan DH, Periasamy M. Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development. Am J Physiol Cell Physiol 262: C614–C620, 1992.
3. Asahi M, McKenna E, Kurzydlowski K, Tada M, MacLennan DH. Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP. J Biol Chem 275: 15034–15038, 2000.
4. Biehn SE, Czymmek KJ, Leavens KF, Karin NJ. Expression of the sarco/endoplasmic Ca2+-ATPase, SERCA1a, in fibroblasts induces the formation of organelle membrane arrays. Exp Cell Res 292: 78–88, 2004.[CrossRef][Web of Science][Medline]
5. Brady ST, Stenoien DL. Axonal transport: molecular, cellular and medical aspects. In: Basic Neurochemistry (6th ed.). New York: Raven, 1997.
6. Cyr JL, Pfister KK, Bloom GS, Slaughter CA, Brady ST. Molecular genetics of kinesin light chains: generation of isoforms by alternative splicing. Proc Natl Acad Sci USA 88: 10114–10118, 1991.
7. De Smedt H, Eggermont JA, Wuytack F, Parys JB, Van den Bosch L, Missiaen L, Verbis J, Casteels R. Isoform switching of the sarco(endo)plasmic reticulum Ca2+ pump during differentiation of BC3H1 myoblasts. J Biol Chem 266: 7092–7095, 1991.
8. Ferrington DA, Yao Q, Squier TC, Bigelow DJ. Comparable levels of Ca-ATPase inhibition by PLB in slow-twitch skeletal and cardiac sarcoplasmic reticulum. Biochemistry 41: 13289–13296, 2002.[CrossRef][Medline]
9. Griffin BA, Adams SR, Jones J, Tsien RY. Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol 327: 565–578, 2000.[Web of Science][Medline]
10. Haghighi K, Kolokathis F, Pater L, Lynch RA, Asahi M, Gramolini AO, Fan GC, Tsiapras D, Hahn HS, Adamopoulos S, Liggett SB, Dorn GW 2nd, MacLennan DH, Kremastinos DT, Kranias EG. Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human. J Clin Invest 111: 869–876, 2003.[CrossRef][Web of Science][Medline]
11. Holland PC, MacLennan DH. Assembly of the sarcoplasmic reticulum. Biosynthesis of the adenosine triphosphatase in rat skeletal muscle cell culture. J Biol Chem 251: 2030–2036, 1976.
12. Jones LR, Cornea RL, Chen Z. Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking. J Biol Chem 277: 28319–28329, 2002.
13. Kaprielian Z, Fambrough DM. Expression of fast and slow isoforms of the Ca2+-ATPase in developing chick skeletal muscle. Dev Biol 124: 490–503, 1987.[CrossRef][Web of Science][Medline]
14. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21: 86–89, 2003.[CrossRef][Web of Science][Medline]
15. Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K. Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci USA 101: 9955–9959, 2004.
16. Kiss E, Brittsan AG, Edes I, Grupp IL, Grupp G, Kranias EG. Thyroid hormone-induced alterations in phospholamban-deficient mouse hearts. Circ Res 8: 608–613, 1998.
17. Lakatta EG, Sollott SJ. Perspectives on mammalian cardiovascular aging: humans to molecules. Comp Biochem Physiol A Mol Integr Physiol 132: 699–721, 2002.[CrossRef][Medline]
18. MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol 4: 566–577, 2003.[CrossRef][Web of Science][Medline]
19. MacLennan DH, Zubrzycka E, Jorgensen AO. Assembly of sarcoplasmic reticulum during muscle development. Curr Top Membr Transp 24: 337–368, 1985.
20. Mahaney JE, Albers RW, Kutchai H, Froehlich JP. Phospholamban inhibits Ca2+ pump oligomerization and intersubunit free energy exchange leading to activation of cardiac muscle SERCA2a. Ann NY Acad Sci 986: 338–340, 2003.[Web of Science][Medline]
21. Meusser B, Hirsch C, Jarosch E, Sommer T. ERAD: the long road to destruction. Nat Cell Biol 7: 66–72, 2005.
22. Murshid A, Presley JF. ER-to-Golgi transport and cytoskeletal interactions in animal cells. Cell Mol Life Sci 61: 133–145, 2004.[CrossRef][Web of Science][Medline]
23. Nori A, Bortoloso E, Frasson F, Valle G, Volpe P. Vesicle budding from endoplasmic reticulum is involved in calsequestrin routing to sarcoplasmic reticulum of skeletal muscles. Biochem J 379: 505–512, 2004.[CrossRef][Web of Science][Medline]
24. Pegg W, Michalak M. Differentiation of sarcoplasmic reticulum during cardiac myogenesis. Am J Physiol Heart Circ Physiol 252: H22–H31, 1987.
25. Pfister KK, Wagner MC, Stenoien DL, Brady ST, Bloom GS. Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultures cells. J Cell Biol 108: 1453–1463, 1989.
26. Sharma P, Patchell VB, Gao Y, Evans JS, Levine BA. Cytoplasmic interactions between phospholamban residues 1–20 and the calcium-activated ATPase of the sarcoplasmic reticulum. Biochem J 355: 699–706, 2001.[Web of Science][Medline]
27. Squier TC, Bigelow DJ. Redox modulation of cellular signaling and metabolism through reversible oxidation of methionine sensors in calcium regulatory proteins. Biochim Biophys Acta 1703: 121–134, 2005.[Medline]
28. Stenoien DL. Characterization and Functional Analyses of the Kinesin Light Chains and Light Chain Isoforms (PhD dissertation). Dallas, TX: University of Texas Southwestern at Dallas, 1996.
29. Stenoien DL, Cummings CJ, Adams HP, Mancini MG, Patel K, DeMartino GN, Marcelli M, Weigel NL, Mancini MA. Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone. Hum Mol Genet 8: 731–741, 1999.
30. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, Mancini MA. FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent. Nat Cell Biol 3: 15–21, 2001.[CrossRef][Web of Science][Medline]
31. Tada M. Calcium cycling proteins of the cardiac sarcoplasmic reticulum. Circ J 67: 729–737, 2003.[CrossRef][Web of Science][Medline]
32. Tuvia S, Buhusi M, Davis L, Reedy M, Bennett V. Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins. J Cell Biol 147: 995–1008, 1999.
33. Vangheluwe P, Tuwa M, Van Den Bergh A, Louch WE, Beullens M, Dode L, Carmeliet P, Kranias E, Herijgers P, Sipido KR, Raeymaekers L, Wuytack FJ. A SERCA2 pump with an increased Ca affinity can lead to severe cardiac hypertrophy, stress intolerance and reduced life span. J Mol Cell Cardiol 41: 308–317, 2006.[CrossRef][Web of Science][Medline]
34. Verboomen H, Wuytack F, De Smedt H, Himpens B, Casteels R. Functional difference between SERCA2a and SERCA2b Ca2+ pumps and their modulation by phospholamban. Biochem J 286: 591–595, 1992.[Web of Science][Medline]
35. Ver Heyen M, Heymans S, Antoons G, Reed T, Periasamy M, Awede B, Lebacq J, Vangheluwe P, Dewerchin M, Collen D, Sipido K, Carmeliet P, Wuytack F. Replacement of the muscle-specific sarcoplasmic reticulum Ca-ATPase isoform SERCA2a by the nonmuscle SERCA2b homologue causes mild concentric hypertrophy and impairs contraction-relaxation in the heart. Circ Res 89: 838–846, 2001.
36. Yao Q, Bevan JL, Weaver RF, Bigelow DJ. Purification of porcine phospholamban expressed in Escherichia coli. Protein Expr Purif 8: 463–468, 1996.[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  J. Barop, H. Sauer, K. Steger, and M. Wimmer Differentiation-dependent PTPIP51 Expression in Human Skeletal Muscle Cell Culture J. Histochem. Cytochem., May 1, 2009; 57(5): 425 - 435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Z. Lin and L. Wang Selective Labeling of Proteins with Chemical Probes in Living Cells Physiology, June 1, 2008; 23(3): 131 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Robia, K. S. Campbell, E. M. Kelly, Z. Hou, D. L. Winters, and D. D. Thomas Forster Transfer Recovery Reveals That Phospholamban Exchanges Slowly From Pentamers but Rapidly From the SERCA Regulatory Complex Circ. Res., November 26, 2007; 101(11): 1123 - 1129. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Avila, C. I. Aguilar, and R. Ramos-Mondragon Sustained CGRP1 receptor stimulation modulates development of EC coupling by cAMP/PKA signalling pathway in mouse skeletal myotubes J. Physiol., October 1, 2007; 584(1): 47 - 57. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
), SERCA1 (
), or SERCA2 (
) at indicated times after placement of myocytes into differentiation medium. C: visualization by immunostaining of cellular distributions of PLB (top) or SERCA1 (bottom) in myoblasts before differentiation (left) or after 3 days in differentiation medium (right). Relative protein abundances from densitometry were normalized to those observed in differentiated myocytes (day 6). PLB, SERCA1a, and SERCA2 were respectively detected using murine monoclonal antibodies 2D12, IIH11, and 2A7-A1 (Affinity BioReagents), and AlexaFluor 594 goat anti-mouse secondary antibodies were used for immunostaining of myocytes.
