Cellular Trafficking of Phospholamban and Formation of Functional
Sarcoplasmic Reticulum during Myocyte Differentiation.
David L. Stenoien, Tatyana V. Knyushko, Monica P. Londono,
Lee K. Opresko, M. Uljana Mayer, Scott T. Brady1, Thomas C. Squier, and Diana J. Bigelow*
Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest
National Laboratory, P.O. Box 999, Richland, WA 99352
and 1Department of Anatomy and Cell Biology Department, University of Illinois at Chicago,
Chicago, IL 60612
Running Header: Vesicle-Guided Trafficking of Phospholamban
Key words: phospholamban, SERCA, differentiation, C2C12 myocytes, vesicle trafficking
Page 1 of 42Articles in PresS. Am J Physiol Cell Physiol (February 7, 2007). doi:10.1152/ajpcell.00523.2006
Copyright © 2007 by the American Physiological Society.
Correspondence should be addressed to: Diana J. Bigelow, Pacific Northwest National
Laboratory, P.O. Box 999, MS P7-56; Richland, WA 99352; email: firstname.lastname@example.org;
Tel: (509) 376-2378; Facs: (509) 376-6767
Page 2 of 42
Phospholamban (PLB) associates with the Ca-ATPase in sarcoplasmic reticulum (SR)
membranes to permit the modulation of contraction in response to beta-adrenergic signaling.
To understand how coordinated changes in the abundance and intracellular trafficking of PLB
and the Ca-ATPase contribute to the maturation of functional muscle, we have measured
changes in abundance, location, and turnover of endogenous and tagged proteins in
myoblasts and during their differentiation. We find that PLB is constitutively expressed in
both myoblasts and differentiated myotubes, whereas abundance increases of the Ca-
ATPase coincide with the formation of differentiated myotubes. We observe that PLB is
primarily present in highly mobile vesicular structures outside the endoplasmic reticulum,
irrespective of the expression of the Ca-ATPase, indicating that PLB targeting is regulated
through vesicle trafficking. Moreover, using pulse chase methods, we observe that in
myoblasts PLB is trafficked through directed transport through the Golgi to the plasma
membrane prior to 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 Ca-ATPase.
Page 3 of 42
Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; BG, O6-benzyl guanine; CFP,
cyan fluorescent protein; DM, differentiation media; DMEM, Dulbececco’s modified Eagle’s
medium; FBS, fetal bovine serum; FRAP, fluorescence recovery after photobleaching; GFP,
green fluorescent protein; HS, horse serum; PLB, phospholamban; SERCA,
sarco/endoplasmic reticulum calcium ATPase; TMR, tetramethylrhodamine; YFP, yellow
Page 4 of 42
The sarcoplasmic reticulum (SR) Ca-ATPase mediates the rate-limiting re-
sequestration of calcium ions from the cytosol to the SR lumen after each contractile event in
muscle. Its association with the inhibitory protein, phospholamban (PLB), provides an
additional level of regulation whereby calcium-transport rates, and the force of contraction,
are directly modulated by beta-adrenergic stimulation by means of the phosphorylation of
PLB through either cAMP- or CaM-dependent kinases (Asahi et al., 2000; Sharma et al.,
2001; MacLennan and Kranias, 2003; Tada, 2003; Squier and Bigelow, 2006). PLB and the
Ca-ATPase reside as an integral membrane protein complex in the SR, where maximal
inhibition of the Ca-ATPase requires an equal stoichiometry of PLB, as is present in co-
expressed or reconstituted vesicles, as well as in vivo in SR isolated from slow-twitch skeletal
muscle (Ferrington et al., 2002; Mahaney et al., 2003). However, in the heart, PLB is
present in a 3-4 fold molar excess relative to the Ca-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 Ca-ATPase is highlighted by variations in PLB-to-Ca-ATPase ratios that correlate with
defective calcium regulation in hyper- and hypo-thyroidism, aging, and the failing heart (Kiss
et al., 1998; Lakatta and Solott, 2002). Furthermore, genetic manipulations have
demonstrated that cellular changes in PLB-to-Ca-ATPase molar ratios can act to
compensate for altered calcium affinities of Ca-ATPase isoforms (Vanghelewe et al., 2006).
The initial formation of PLB-Ca-ATPase interactions in the developing heart is
coincident with progressively decreasing stoichiometries of total expressed PLB relative to
Ca-ATPase as differentiation to mature myocytes occurs (Pegg and Michalek, 1987; Arai et
al., 1992). PLB is expressed early in the undifferentiated myocyte, whereas up-regulation of
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the Ca-ATPase occurs coincident with differentiation and the formation of the SR membrane
which is a specialized region of the 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 Ca-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 have 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 Ca-ATPase, we find that both endogenous and transfected
Ca-ATPase exhibits 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 prior to their degradation. Following differentiation, PLB colocalizes
with the Ca-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 suggests a possible role for PLB in the plasma membrane associated with
Page 6 of 42
Material and Methods
Reagents. The GFP-SERCA1a vector, coding for the chicken SERCA1a sequence, (Biehn,
2004) was a kind gift from Norman J. Karin. GFP-PLB was made by isolating a
BamHI/EcoRI fragment from the PLB-pGEX2T vector (Yao, 1996) containing the porcine
PLB sequence and inserting it into pEGFP-C1 (Clontech; Mountain View, CA) digested with
BglII/EcoRI. Cyanofluorescent protein (CFP) and yellow fluorescent protein (YFP) versions of
these vectors were made by replacing the AgeI/BsrG1 fragment from the GFP vectors with
the corresponding fragments from the pECFP-C1 and pEYFP-C1 vectors (Clontech).
FlAsH·EDT2 was synthesized as previously described (Mayer et al., 2005)
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 using the Age1 and BsrG1
restriction sites to create the vector pAGT-C1 containing the same multi-cloning 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) 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
Page 7 of 42
sequence encoding the amino acids MAEAAAREACCPGCCARAR. The two oligos were
annealed, 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 above 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 (Gibco),
penicillin/streptomycin, 1 mM sodium pyruvate, and 4 mM L-glutamine. For differentiation
experiments, the growth medium, described above, was replaced with differentiation medium
(DM) consisting of DMEM, 2% horse serum, penicillin/streptomycin, 1mM 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% NP-40 (w/v), 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 16,000xg) and the protein concentration in the
lysates was determined prior to electrophoretic separation. SDS-PAGE was performed using
7.5, 15 or 4-12% polyacrylamide gels. Proteins were transferred to a PVDF membrane using
a semi-dry transfer cell (Bio-Rad) for 30 min at 25V for SERCA1 and SERCA2 identification,
and for 15 min at 20V for PLB identification, in buffer containing 25 mM TRIS, 192 mM
glycine, 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 (Jones et al., 2002), with dilution 1:10,000, SERCA1
Page 8 of 42
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 ECL chemiluminescence (Amersham; Piscataway, NJ). To detect
endogenous proteins in myocytes, immunolabeling was carried out as previously described
(Stenoien et al, 1999) 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 (FRAP).
Live-cell microscopy was performed on cells transiently transfected with GFP-, CFP-, or YFP-
or tagged PLB or SERCA1a as indicated in the figure legends. Cells were either fixed for co-
labeling 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, cells
were incubated in media containing 4 µM BG-505, BG-DAF, or BG-tetra methyl rhodamine
(BG-TMR) star for 30 minutes followed by a 15 minute wash in media 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 media before relabeling with BG-505. BG-
505 was visualized using fluorescein filter sets while BG-TMR was imaged using rhodamine
filter sets. Labeling TC-PLB expressing cells involved incubation of cells for 30 minutes in
HEPES buffered saline (HBS) containing 1 µM FlAsH·EDT2 reagent. Cells were washed with
HBS, incubated 5 minutes in HBS containing 5 mM EDT to reduce non-specific background
and washed 2X in HBS before replacement of tissue culture media and imaging of live cells.
Page 9 of 42
The supplemental movie showing movement of AGT-PLB was acquired using Metamorph
software (Universal Imaging Corp., Downington, PA) to acquire a stack of timelapse images
and saved in Quicktime format.
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 percent laser power for a total
duration of ~1 sec. 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 figures assembled using Adobe
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 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-
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
previously described (Pfister et al., 1989). The monoclonal antibody (KLC-2B) recognizing a
subset of kinesin light chain 1 alternatively spliced isoforms (KLC-B and KLC-C; Cyr et al.,
1991) was generated by immunizing mice with a peptide (MRKMKLGLVK) found exclusively
in these kinesin light chainschain1 isoforms and producing antibodies as described
Page 10 of 42
previously (Pfister et al., 1989). These antibodies have been extensively characterized and
recognize a subset of native kinesin light chains and bacterial expressed kinesin light chains
B and C, but not A (Stenoien, 1996). 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 NICHD 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 manufacturers
instructions. Benzyl guanine (BG)-505, BG-diacetylfluorescein (BG-DAF), and BG-TMR were
obtained from Open Biosystems (Huntsville, AL). All cell culture media, unless otherwise
stated, was obtained from Gibco Laboratories (Grand Island, NY).
Page 11 of 42
Differential Abundance of PLB and Ca-ATPase Proteins During Myocyte
Differentiation. C2C12 myocytes provide an accurate model of developing muscle as they
can be readily induced to differentiate. PLB expression is essentially constitutive throughout
differentiation while that of the Ca-ATPase (the SERCA1 and SERCA2 isoforms) is up-
regulated coincident with differentiation into myotubes. This differential expression is
illustrated by Western blot analysis (Figure 1A). PLB is already present in undifferentiated
myoblasts (day 0), whereas both fast-twitch (SERCA1) and slow-twitch (SERCA2) isoforms
of the Ca-ATPase are present at low or undetectable levels in these cells. Of note,
SERCA2b has been observed to be present at low levels in the ER of myoblasts, whereas its
splice isoform SERCA2a is predominant in differentiated myocytes; both SERCA2 isoforms
are detected by this antibody (De Smedt, et al., 1991; Arai et al., 1992). To determine how
differentiation affects the relative expression of these proteins, C2C12 cells were allowed to
reach ~90% confluency in growth media (DMEM with 10% FBS) prior to its replacement with
differentiation media (DMEM with 2% HS) for 1 to 7 days. As shown in Figure 1B,
differentiation has no significant effect on total PLB expression levels, but both SERCA1a
and SERCA2 show a linear increase in abundance as cells differentiate. This increase in
SERCA abundance coincides 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 is 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 Ca-ATPase is a general feature of
cultured myocytes and this pattern mirrors the same changes in relative abundances of PLB
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and the Ca-ATPase observed in the developing heart (Pegg & Michalak, 1987; Arai et al.,
Cellular Localization of Endogenous PLB and SERCA2a. In adult myocytes, the
PLB-SERCA complex of integral membrane proteins resides in the SR membrane, which
develops during differentiation as a specialized and finally extensive region of the
endoplasmic reticulum (ER) (MacLennan et al. 1985). To determine the cellular distribution
of PLB as myocytes differentiate, C2C12 myocytes were fixed and immunostained for PLB in
comparison with SERCA1. Myoblasts immunostained for PLB do not show a reticular
distribution, but rather a punctate staining, indicative of vesicles that are distinct from the ER
(Figure 1C; upper left). Only in myotubes does PLB staining show a reticular staining
consistent with ER or SR localization (Figure 1C; upper right). No detectable SERCA1
immunostaining is observed in undifferentiated myoblasts (Figure 1C; lower left), consistent
with the absence of total SERCA protein assayed by Western blot (Figure 1B). However, in
myocytes in which the differentiation program has been activated, SERCA1 is detected and
exhibits a reticular distribution consistent with SR localization (Figure 1C; lower right). These
observations suggest the requirement for altered trafficking of PLB from its localization in
vesicles to the SR membrane for the formation of PLB-SERCA interactions during
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 Karin and
coworkers (Biehn et al., 2004). The distribution of GFP-PLB (Figure 2, upper left), like that
of endogenous PLB (Figure 1), exhibits a punctate pattern that extends to the edge of the
Page 13 of 42
cell and localizes to regions of the plasma membrane and cellular extensions in many cells.
On the other hand, in myoblasts GFP-tagged SERCA1a (Figure 2; lower left) is constrained
within, but evenly distributed throughout the endoplasm and is observed to be reticular in
many regions. This reticular intracellular distribution is similar to that expected for wildtype
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 (Biehn et al., 2004).
Immunolabeling of GFP transfected cells with anti-PLB or anti-SERCA1a antibodies
shows that the GFP localization pattern coincides with the immunolocalization pattern (data
not shown). In the case of GFP-SERCA1a, the expressed sequence is from chicken,
permitting the use of an antibody specific for avian SERCA1a to specifically detect only the
transfected protein (Kaprielian and Fambrough, 1987). Thus, the identical patterns of
localization of endogenous and GFP-tagged PLB indicate both that the GFP-tag does not
affect normal protein distribution in the myoblast and that the fixation required for
immunolabeling of endogenous PLB does not produce artifactual staining patterns.
In order to further compare the intracellular trafficking and turnover of PLB and
SERCA, stable cell lines were created expressing PLB and SERCA, each as fusion proteins
with O6-alkylguanine-DNA alkyltransferase (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 suitable for live-cell imaging (Keppler et al., 2003; 2004). These stable
myocyte cell lines were amenable to differentiation into myotubes in which the levels of
expressed AGT-PLB and AGT-SERCA protein were approximately 50% of that of
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endogenous (wildtype) PLB and SERCA (data not shown). As shown in the center panels of
Figure 2, C2C12 myoblasts expressing AGT-PLB and labeled with a BG fluorescein
derivative, BG-505, show the same punctate distribution as endogenous or GFP-tagged 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 (21kDa) tags relative to
the smaller PLB (6 kDa) may produce artifactual aggregation, a PLB construct was
genetically engineered with an N-terminal sequence containing a small tetracysteine motif
suitable for labeling with the cell-permeable FlAsH (<700 Da; Griffin et al., 2000). This
tetracysteine-tagged PLB (C4-PLB) expressed in C2C12 myoblasts and labeled with FlAsH
also exhibits a punctate fluorescence pattern (Figure 2; right panel). Thus, the punctate
distribution of PLB in undifferentiated myoblasts consistently observed for wildtype 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 fluorescence recovery after photobleaching (FRAP), since rates of lateral
protein mobility correlate with their cell locale. As shown in panels Figure 3A, when a region
of a myoblast expressing GFP-SERCA1a is photobleached, a gradual, uniform recovery of
fluorescence is observed. The half-life of recovery is ~20 seconds, significantly slower than
the half-life of freely diffusing GFP (~0.3 s; Stenoien et al, 2001). 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 is
Page 15 of 42
observed (Figure 3B). First, the recovery occurs more slowly requiring several minutes for
complete recovery of the fluorescence. Second, the GFP-PLB enters the bleached area as
discrete, subcellular structures that suggest recovery is due to transport of GFP-PLB
containing vesicles into the bleached zone (see magnified images in Figure 3C). Thus,
these very different rates of lateral diffusion of SERCA as compared with PLB are
incompatible with their co-localization indicating their localization to distinct compartments,
i.e., ER and vesicles, respectively, in undifferentiated myocytes.
PLB-Containing Vesicle Movements are Characteristic of Microtubule-Based
Transport. To track the movement of individual PLB -containing vesicles, we performed live
cell imaging of both GFP- and AGT-tagged PLB, both of which gave equivalent results.
Panels D in Figure 3 show a magnified portion of a live cell stably expressing AGT-PLB and
labeled with the fluorescein dye, BG-505; many of the PLB containing vesicles undergo rapid
bidirectional movement in the cell. Further expansion of a portion of this cell in the
subsequent panels show one AGT-PLB-containing vesicle (large arrow) that undergoes
bidirectional transport over time, on what appears to be a linear track, relative to two other
vesicles that remain more stationary (small arrow) and therefore serve as landmarks. The
movement in both directions appears to occur by pulling as the vesicle is stretched in the
direction of the movement followed by a rapid movement of the bulk of the vesicle in the
same direction. The movement can be better appreciated in the movie included as
supplemental data (Figure S1). This bidirectional movement is characteristic of microtubule-
based transport mediated by kinesins and cytoplasmic dynein and further suggests that the
directed transport of PLB in vesicles occurs in myoblasts (Brady and Stenoien, 1997).
Page 16 of 42
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 is 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 (Figure 4A). As
illustrated in the top panel, the fluorescence associated with labeled PLB has disappeared
within 24 hours and is accompanied by a noticeable time-dependent change in the
distribution of the labeled PLB within the cell. Specifically, fluorescence at the plasma
membrane disappears relatively early on and, over time, the fluorescence becomes less
intense, but more centrally localized around the nucleus, suggesting that recently
synthesized PLB is preferentially targeted to the perinuclear region, whereas at later times
after synthesis PLB is localized at the plasma membrane. In contrast, fluorescently labeled
AGT-SERCA exhibits a relatively stable reticular distribution over the timecourse of the
experiment as its fluorescence intensity diminishes, consistent with the synthesis and
retention of AGT-SERCA as an integral ER membrane protein. Moreover, fluorescence
associated with SERCA persists for over 24 hours (Figure 4A), consistent with the 30 h half-
life for SERCA1 previously measured with radioisotopes in chick myocytes (Holland and
MacLennan, 1976). The more rapid loss of fluorescence associated with AGT-PLB in
Page 17 of 42
comparison with that of AGT-SERCA suggest its shorter protein half-life than that of SERCA;
to our knowledge, protein turnover of PLB has not been previously measured.
Additional kinetic resolution of cellular trafficking was obtained through a dual color
pulse chase experiment, permitting the measurement of the cellular localization of newly
synthesized proteins (Figure 4B). In this case, existing AGT-PLB or AGT-SERCA1a was
labeled with BG-tetramethylrhodamine (TMR; in red); after an additional 5 hours in culture,
newly synthesized proteins were labeled with BG-505 (green). In the case of PLB (Figure
4B, upper panels), both the pre-existing protein (red) and newly synthesized protein (green)
have similar cellular distributions, with the exception that there are elevated levels of newly
synthesized protein, as evidenced by the concentration of green label, in a perinuclear region
(denoted by the arrow). There is significant overlap in the distribution of the red and green
labeled PLB in other regions of the cell. Similar experiments performed with AGT-SERCA
show that both existing and newly synthesized SERCA are predominantly localized in the ER
(Figure 4B). Thus, in contrast to the redistribution of PLB observed as a function of its time
after synthesis in the myoblast, SERCA appears to be synthesized at or near its site of final
and processed rapidly with a short residence time in the cell without evidence of cellular
trafficking.Golgi and longer residence in cytoplasmic compartments allowing it to equilibrate
in all pools within the time window used in these studies. These data indicate that SERCA is
trafficked independent of PLB.
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 SERCA2a as it becomes available in the
Page 18 of 42
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 Figure
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 does
not affect the localization of PLB in myoblasts. In this case, untagged SERCA2a was utilized
in order to avoid any potential interference of a tag with PLB-SERCA2a interactions (Figure
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 colocalized
We next addressed whether changes in PLB localization correlate with the initiation of
differentiation for individual myocytes. Since differentiated myocytes are not amenable to
transfection, the initial analysis involved transient transfection with YFP-PLB and CFP-
SERCA1a and analysis after 2 days in differentiation media, resulting in a mixed population
of both myoblasts and myocytes in the early stages of differentiation. Under these
conditions, most of the transfected cells remain as myoblasts which do not express MHC,
while both PLB and SERCA retain their unique distributions (Figure 5C). Moreover, in the
small number of cells that had begun to differentiate as judged by both MHC expression and
multi-nucleation, YFP-PLB develops a similar localization pattern as CFP-SERCA1a (Figure
5D). Using our stable cell lines expressing AGT-PLB and AGT-SERCA1a, we were able to
extend our analysis to examine later stages of differentiation. After 4 days of differentiation,
AGT-PLB loses its punctuate distribution (Figure 5E) and adopts a similar distribution as
Page 19 of 42
AGT-SERCA1a (Figure 5F). Together these results suggest that differentiation must occur
before PLB colocalizes with SERCA isoforms in SR membranes.
Co-Localization 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 co-localization experiments with cell compartment specific dyes and antibodies to
identify the cellular compartment(s) associated with PLB trafficking. As shown in Figure 6A,
a concentration of AGT-PLB is observed in the region of the Golgi, as evidenced by co-
localization of a subset of PLB in the perinuclear region stained with an antibody to the Golgi
resident protein, golgin. In contrast, there is no significant co-localization 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 co-localization of AGT-PLB (shown labeled with BG-
505 in green) and an RFP-endosome marker, i.e., the RhoB GTPase (Figure 6B and C).
We find extensive co-localization of PLB with the labeled endosomes, as highlighted in the
enlarged image that shows many vesicles containing both PLB and the RFP-Endo (Figure
As a further demonstration that PLB localizes to transported vesicles, we performed
co-localization experiments with antibodies recognizing the vesicle transport motor, kinesin.
Shown in Figure 6D is an AGT-PLB expressing cell labeled with an antibody (KLC-2B)
recognizing a subset of kinesin light chain-1 isoforms generated by alternative splicing (Cyr
et al, 1991). This figure shows that regions near the plasma membrane containing AGT-PLB
also contain elevated levels of kinesin light chains suggesting that these membrane domains
Page 20 of 42
are targeted by actively transported vesicles. Similar results were obtained with an antibody
(H2; Pfister et al, 1989) recognizing kinesin heavy chains (Figure 6E). These colocalization
results support the directed transport of PLB containing vesicles in undifferentiated
myoblasts, from its site of synthesis near the perinuclear region with movement to the plasma
membrane through the Golgi prior to internalization and subsequent degradation of PLB by
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Changes in relative abundances of PLB and the SR Ca-ATPase occurring in heart
disease and other patho-physiological states suggest the importance of understanding the
trafficking pathways associated with the biosynthesis of functional SR membranes. Using
both wildtype and fluorescent-tagged protein expressing C2C12 myocytes, we find striking
differences in the abundance, cellular localization, and trafficking of PLB relative to the Ca-
ATPase in myoblasts prior to their colocalization in the differentiated myotube. In particular,
in myoblasts, a punctate staining pattern for PLB is consistently observed that is distinct from
the reticular staining of expressed Ca-ATPase; this pattern suggests a vesicular localization
for PLB (Figures 1-6). The very different rates of fluorescence recovery after photobleaching
for PLB and the Ca-ATPase in the myoblast lends additional support for their distinct cellular
compartments (Figure 3). From live cell imaging the bidirectional character of movement of
the PLB-associated points of fluorescence suggests the transport of vesicles along
microtubules as does the colocalization with the motor protein, kinesin (Figures 3 and 6).
Pulse chase experiments that permit the fluorescent labeling of a transient population of
proteins indicate that recently synthesized PLB is more highly concentrated in a perinuclear
locale consistent with synthesis within the rough ER, whereas at later times after its synthesis
the distribution of PLB extends to the periphery of the cell (Figures 2 and 4). The early loss
of fluorescence of labeled AGT-PLB at the plasma membrane and the colocalization of PLB
with RhoB and Golgin suggests movement of PLB vesicles outward towards the plasma
membrane through the Golgi apparatus where PLB becomes incorporated into endosomes
and finally degraded. In this regard, a truncation mutant of PLB (PLB1-39) that leads to a
lethal cardiomyopathy in humans exhibits accumulation at the plasma membrane when
Page 22 of 42
expressed in tissue culture and is less stable than the wild type protein (Haghighi et al,
2003). This altered localization of PLB could be due to its failure to be endocytosed at the
plasma membrane, thereby disrupting the normal trafficking cycle, that is proposed here.
Of note, while 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 (Figures 1-6). This observation is
consistent with the expression of SERCA2b in myoblasts, and prior observations regarding
the ability of PLB to regulate SERCA2b when coexpressed in cells and in genetically altered
mice (Verboomen et al., 1992). Thus, PLB may have a dual role in the myoblast, one, to
regulate SERCA2b in the ER, and the 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 is 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 does the bulk of PLB colocalize with the Ca-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 Ca-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 Ca-ATPase,
resides primarily in the SR after differentiation. However, it cannot be ruled out that the
Page 23 of 42
extensive proliferation of SR in myotubes masks ready detection of a minor vesicular
population of PLB that trafficks from ER to plasma membrane (Figure 7).
Based on the extensive knowledge gained in recent years regarding trafficking
pathways for plasma membrane and secreted proteins, it is known that these proteins are
synthesized in the ER and exported at ER exit sites via COPII (coat protein complex II)
vesicles and transported by microtubules to the ER-Golgi intermediate complex (ERGIC)
where sorting occurs before trafficking to their final cellular destination (Murshid and Presley,
2004). Thus, the co-localization of PLB vesicles with the motor protein, kinesin, and the
vesicle movements observed by live cell imaging that are characteristic of microtubule
transport strongly supports the directed trafficking of PLB (Figures 2 and 6). Moreover, this
result rules out a significant involvement of the alternative pathway for ER exit, i.e., the
retrograde transport of misfolded proteins that are routed to the cytoplasm for proteasomal
degradation. The proteasome is located at the cytoplasmic face of the ER membrane for
retrograde protein exit and directed transport mechanisms are not involved (Meusser et al.,
2005). Instead, the colocalization of PLB with endosomes (Figure 6) suggests that PLB
degradation involves lysosomal degradation, and is reminiscent of the mechanisms by which
receptor abundance at the cell surface is regulated. Resident ER proteins that do escape
from the ER in COPII vesicles are retrieved and recycled via specific carboxy-terminal ER
retention sequences such as the dilysine (-KKXX) motif (in type I integral membrane
proteins) and KDEL (in ER lumenal proteins); these motifs are not found within the PLB
sequence (Murshid and Presley, 2004). Alternatively, ER proteins may be selectively
retained through binding to other ER proteins; however, such a mechanism involving binding
to SERCA2a, the normal binding partner of PLB, can be excluded. Thus, the trafficking of
Page 24 of 42
PLB in myoblasts would appear to be consistent with classic secretory routes, and suggests
a novel function for PLB in the myoblast.
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 (Nori et al., 2004). Similarly,
SERCA in the heart has been suggested to be routed to SR via vesicles by an ankyrin-B
assisted protein sorting mechanism (Tuvia, 1999). 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, which is distinct from that
associated with the Ca-ATPase, its interaction partner in the mature myocyte. These
observations suggest possible new roles for PLB in the myoblast, and highlights the
important cellular regulation associated with the trafficking of PLB in the formation of fully
functional SR membranes in which the Ca-ATPase is under beta-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
Page 25 of 42
Acknowledgements: The research described in this paper was conducted under the LDRD
Program at the Pacific Northwest National Laboratory: a multi-program national laboratory
operated by Battelle for the U.S. Department of Energy under Contract DE-AC06-
76RL01830. Portions of the work were also supported by NIH grants HLB064031 (TCS) and
Page 26 of 42
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Figure 1: Abundance of Endogenous PLB and SERCA During Myocyte Differentiation.
Immunoblots (A) and measured protein abundances (B) for PLB (triangles), SERCA1 (open
circles), or SERCA2 (closed circles) at indicated times after placement of myocytes into
differentiation media. Visualization by immunostaining of cellular distributions of (C) PLB
(upper panels) or SERCA1 (lower panels) in myoblasts before differentiation (left panels) or
after 3 days in differentiation media right panels. 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, for immunostaining of myocytes,
AlexaFluor 594 goat anti-mouse secondary antibodies.
Figure 2: Localization of PLB and SERCA in Undifferentiated Myoblasts. Fluorescence
of cells expressing either (A) GFP-PLB (upper left panel) or GFP-SERCA1a (lower left
panel); (B) AGT-PLB (upper center panel) or AGT-SERCA1a (lower center panel) following
labeling with BG-DAF substrate; or (C) tetracysteine-PLB labeled with FlAsH (upper right
panel); the specificity of this pattern over any background is demonstrated by the lower
FlAsH fluorescence observed in untransfected cells (lower right panel). Myocyte transfection
and labeling were performed as described in Materials and Methods.
Figure 3: FRAP and Live Cell Imaging of PLB and SERCA. Fluorescence of GFP-
SERCA1a (A) or GFP-PLB (B) is shown at the indicated times following photobleaching the
area within the rectangle. GFP-PLB fluorescence recovers by movement of unbleached
Page 33 of 42
vesicles into the bleached area as seen in the enlarged view in panel C. This movement is
highlighted in the complementary live cell imaging of an individual PLB-containing vesicle
(large arrow) relative to an immobile vesicle (small arrow) at 10 s intervals (D). Bar = 10 um.
A movie showing the movement of these vesicles can be seen in the Supplemental Data.
Figure 4: Kinetics of Degradation and Biosynthesis of AGT-tagged PLB and SERCA1.
A. AGT-PLB (upper row) or AGT-SERCA1a (lower row) expressing myoblasts were directly
imaged at indicated times following labeling with BG-DAF. B: Alternatively, cells were first
labeled with BG-TMR (red), washed and incubated in media for 5 h prior to labeling with BG-
DAF (green) (B), permitting the resolution of newly synthesized proteins. Overlay (right panel
in B) shows co-localization of newly synthesized (green) and existing (red) proteins.
Figure 5: Cellular Differentiation and Localization of PLB and SERCA. In myoblasts at
day 0 of differentiation (D0), YFP-PLB (A) or AGT-PLB (B) have distinct localizations relative
to either coexpressed CFP-SERCA1a (A) or coexpressed SERCA2a imaged using
immunofluorescence. The corresponding merged images are shown in the right panels. Co-
localization of YFP-PLB and CFP-SERCA coincident with differentiation and MHC expression
(D) relative to undifferentiated cells (C) is apparent in fused images (right panels) following
two days of growth in differentiation media (D2). Images of differentiated myotubes at day 4
of differentiation (D4) expressing AGT-tagged PLB (E) or AGT-SERCA1a (F) relative to
endogenous MHC imaged using immunofluorescence are shown.
Page 34 of 42
Figure 6: Co-localization of PLB with membrane markers. A. Immunostaining of AGT-
PLB cells with an antibody to golgin 97, reveals some PLB localizes to regions of the cell
containing the Golgi apparatus. In these cells, the Golgi was labeled with a primary
monoclonal antibody to golgin and Alexa Fluor 595 goat anti-mouse secondary antibodies.
B. Shown is a cell expressing both AGT-PLB and RFP-Endo. Some overlap is observed
between the two markers indicating some of the PLB vesicles are endosomes. The extent of
overlap is more apparent in the magnified region of the cell (C). AGT-PLB colocalizes with
kinesin light chain (KLC; D) and kinesin heavy chain (KHC; E) antibodies. Bar = 10 µm.
Figure 7: Model of PLB Possible trafficking routes in myocytes. Newly synthesized PLB
is rapidly transported through the Golgi to the plasma membrane. After endosomal
internalization, PLB is trafficked to lysosomes (1) for degradation in myoblasts or to the SR
(2) to regulate SERCAs in differentiated cells. Alternatively, both PLB and SERCAs are
directly transported from the ER to the SR (3) as the SR is formed during differentiation.
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