Neurite Outgrowth of Mature Retinal Ganglion Cells and
PC12 Cells Requires Activity of CK1d and CK1e
Joachim Bischof1, Adrienne Mu ¨ller2, Miriam Fa ¨nder2, Uwe Knippschild1*., Dietmar Fischer2,3*.
1Department of General, Visceral and Transplantation Surgery, University of Ulm, Ulm, Germany, 2Department of Experimental Neurology, University of Ulm, Ulm,
Germany, 3Department of Experimental Neurology, University of Du ¨sseldorf, Du ¨sseldorf, Germany
Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little
neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI)
enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1d and CK1e
activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In
both cell types CK1d and e were localized in granular particles aligned at microtubules in neurites and growth cones.
Although LI treatment did not measurably affect the expression of CK1d and e, it significantly elevated the specific kinase
activity in the retina. Similarly, CK1d/e specific kinase activity was also elevated in NGF treated PC12 cells compared with
untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1d C-terminal targeting
kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of
CK1d and e markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration
dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth.
Citation: Bischof J, Mu ¨ller A, Fa ¨nder M, Knippschild U, Fischer D (2011) Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of
CK1d and CK1e. PLoS ONE 6(6): e20857. doi:10.1371/journal.pone.0020857
Editor: Irina Agoulnik, Florida International University, United States of America
Received March 8, 2011; Accepted May 10, 2011; Published June 16, 2011
Copyright: ? 2011 Bischof et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work in the lab of Uwe Knippschild is supported by the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung (10-2237-KN3 and 108489). Work in the lab of
Dietmar Fischer is supported by funding of the University of Ulm. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (UK); email@example.com (DF)
. These authors contributed equally to this work.
Neurons of the central nervous system (CNS) are normally
unable to regenerate injured axons. This regenerative failure
severely limitsthe chances of recovery after traumatic injuries in the
CNS, stroke and in certain neurodegenerative diseases. Reasons for
the failure in axonal regeneration are partially due to the
insufficient intrinsic capability of adult neurons to regrow axons
and to inhibitory factors associated with CNS myelin and glial scar
formation [1–5]. Mature retinal ganglion cells (RGCs) are typical
CNS neurons and possess only weak intrinsic potential to regrow
injured axons. However, RGCs are switched into a robust
regenerative state when b/c-crystallins are released from an injured
lens [6–8]. In this state mature RGCs extend axons in culture at
higher growth rates, and regenerate lengthy axons into an injured
optic nerve in vivo [7–10]. Glial derived ciliary neurotrophic factor
the essential mediators of these effects [6,11–16]. However, the
molecular processes and regulatory proteins involved in the
rearrangement of the cytoskeleton and the regulation of neurite
growth in mature RGCs are still poorly understood. Several kinases
such as p38 MAPK, ROCK, PKC and PI3K have been identified
to regulate axon growth cone stability and guidance [17–22].
Studies using RNA interference based screening suggested that
approximately 8–9% of the human kinome are involved in
promoting or inhibiting neurite outgrowth .
Members of the CK1 family comprise a group of ubiquitously
expressed second-messenger independent monomeric serine/
threonine specific kinases. In mammals seven isoforms (namely
CK1a, b, c1–3, d and e) and their various splice variants have been
described. All CK1 isoforms are highly conserved within their
kinase domains, but differ significantly in the length and primary
structure of their non-catalytic N-terminal (9–76 aa) and C-
terminal (from 24 aa up to more than 200 aa) domains. Within the
cell the constitutive phosphotransferase activity of CK1 isoforms is
tightly controlled by autophosphorylation, dephosphorylation,
proteolytic cleavage and localization to different subcellular
compartments [24,25]. CK1 family members are able to modulate
the activity of key regulator proteins involved in several cellular
processes such as cell differentiation [26–31], proliferation,
apoptosis [32–36], circadian rhythm , chromosomal segrega-
tion [38–41] and vesicle transport [39,40,42]. CK1d and CK1e,
which share 97% homology within their kinase domains and still
exhibit 53% homology within their C-terminal regulatory
domains, are able to complement the functions of the CK1
homolog Hrr25 in Saccharomyces cerevisiae . Moreover, they
exhibit partially overlapping functions in mammals. Both isoforms
are highly expressed in the hypophysis, the peripheral nervous
system, and the central nervous system [44,45] and are involved in
regulating circadian rhythm . CK1d has also been reported to
regulate dynamics of the cytoskeleton [39,47–50], which is also
essential for axonal growth.
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Here, we show that CK1d and e are expressed in the growth
cones of RGCs and PC12 cells and provide evidence that CK1d
and e activity is essential for neurite growth and extension.
Analysis of the expression of CK1d and e in the adult
retina and PC12 cells
In previous reports we have shown a low to moderate
expression of CK1d and CK1e in the inner nuclear layer (INL)
of rat retina and a significantly stronger staining in bIII-tubulin-
positive RGCs [44,45]. In order to test whether CK1d and e
expression is altered in injured RGCs or when these neurons enter
into a regenerative state adult rats were subjected either to an optic
nerve cut (ONC) or ONC+lens injury (LI). Neither western blot
analysis (Fig. 1), quantitative real-time PCR nor immunohisto-
chemical analysis (data not shown) revealed notable changes in
retinal CK1d and e expression after ONC or ONC+LI compared
with untreated controls, suggesting that expression of CK1d and e
was neither altered in injured nor regenerating RGCs compared
with naı ¨ve RGCs.
To further explore the localization of CK1d and e expression in
RGCs, we prepared dissociated retinal cell cultures 5 days after
ONC+LI. Such in vivo pretreated RGCs show spontaneous
outgrowth of neurites in culture [15,51–53]. After 48 h in culture
cells were fixed for immunofluorescence staining. bIII-tubulin
positive RGCs revealed a granular staining pattern of CK1d and e
along microtubules in the shaft and in the peripheral zones of the
growth cones (Fig. 2 A, B). CK1d and e were also found to be
distributed in the soma of RGCs (Fig. 2 A, B, vertical panel).
PC12 cells are a commonly used model for studying the
molecular mechanisms underlying neurite outgrowth. Therefore
we investigated the expression of CK1d and e in these cells after
neurite outgrowth stimulation by nerve growth factor (NGF). To
this end, protein lysates of PC12 cells were prepared from
untreated control cells and 1, 2, 4, 8, 24 and 48 h after adding
NGF to cell cultures. As determined by western blot analysis and
subsequent densitometric evaluation CK1d expression slightly and
transiently increased after 4 h (Fig. 3 A, B). After 8 h the
expression returned again to basal levels. Expression of CK1e
significantly decreased 4 and 8 h after NGF stimulation and
returned to basal levels after 24 h (Fig. 3 A, B). Immunofluores-
cence analysis of these cells also showed a distribution of CK1d
and e in the perinuclear area, in the neurite and the growth cone
(Fig. 3 C–F).
CK1d/e kinase activity is increased in differentiating PC12
cells and in the adult retina after LI
Although NGF-stimulation of PC12 cells did not affect
expression levels of CK1d and CK1e we speculated whether it
may change the activity of these kinases. This possibility was tested
by measuring the specific activity of CK1d and e in cell lysate
fractions of either untreated or NGF stimulated PC12 cells. One
representative result of two independent experiments is shown in
Fig. 4 A. GST-p531–64(FP267) is a well known substrate for CK1
with several potential phosphorylation sites . FP267 was
subjected to phosphorylation by aliquots of fractionated protein
lysate as described previously . Three major kinase peaks
eluting between 130–180 mM NaCl, 200–220 mM NaCl, and
220–250 mM NaCl were detected in lysates of untreated cells
(Fig. 4 A). Exposure to NGF for 24 h was followed by a 3-fold
increase of the kinase activity in the fraction of the third kinase
peak (220–250 mM NaCl) (Fig. 4 A). As shown in Fig. 4 B the
presence of IC261, specifically inhibiting CK1d and e in the
micromolar range, compromised kinase activity of lysate fraction
21 (corresponding to the third major kinase peak), confirming that
this fraction mainly represented CK1 activity.
Similar observations were made when kinase activity was
measured in fractionated retina protein lysates. No kinase peak
was detected in fractionated untreated control lysates using FP267
as substrate (Fig. 4 C). In contrast, ONC+LI treatment induced an
increase in kinase activity eluting between 450–475 mM NaCl
(fraction 38; Fig. 4 C). Again, the presence of CK1 activity in
fraction 38 was confirmed by inhibition of the kinase activity by
IC261 (Fig. 4 D).
NGF treatment changes the activity of cellular CK1d
targeting kinases in PC12 cells
The activity of CK1d and CK1e is modulated by autophos-
phorylation and by phosphorylation by cellular kinases within the
regulatory C-terminal domain [56–58]. Thus, changes in the
activity of C-terminal targeting kinases upon NGF stimulation of
PC12 cells may regulate CK1 activity. To determine the activity of
CK1d C-terminal targeting kinases each fraction of separated
protein extracts derived from PC12 cells with or without NGF
treatment was used as a source of enzyme. The CK1d C-terminal
fragment GST-CK1d305–375was used as substrate harboring
potential phosphorylation sites for various cellular kinases . As
shown in Fig. 5 the major kinase activity (eluting at 340–410 mM
NaCl) was reduced by approximately 50% in PC12 cells treated
with NGF for 24 h compared to that measured in untreated
controls. These results point to the possibility that NGF increased
the activity of CK1d indirectly by reducing the activity of C-
terminal targeting kinases.
CK1-specific inhibitors abolish neurite outgrowth of
primary RGCs and PC12 cells
Since CK1d and CK1e proteins were located in growth cones of
RGCs as well as PC12 cells and their kinase activity was increased
in growth-stimulated cells, we supposed that CK1d/e activity may
be involved in neurite outgrowth. To test this possibility we
cultured NGF differentiated PC12 cells either in the absence or
presence of the CK1-specific inhibitors CKI-7 (50 and 200 mM) or
IC261 (0.5, 1.5 and 50 mM). The presence of both inhibitors
significantly compromised NGF induced neurite outgrowth in a
concentration dependent manner compared with NGF treated
PC12 control cells (Fig. 6 A), but did not affect the survival of
PC12 cells in culture (data not shown). In primary RGCs that were
cultured 5 days after ONC+LI in the presence of 50 mM CKI-7
Figure 1. Expression of CK1d and e in the retina. Expression of
CK1d and e in naı ¨ve and regenerating retina. Similar levels of CK1d and e
expression in retinal lysates of untreated animals (C) and rats subjected
to optic nerve cut (ONC), lens injury (LI) or ONC+LI were detected by
western blot analysis using the CK1d specific antibody 128A and the
CK1e specific antibody #610446. Detection of b-actin on stripped
membranes verified loading of same protein amounts.
CK1d/e Activity Is Required for Neurite Outgrowth
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for 24 h, neurite outgrowth was decreased by 86% compared with
controls (Fig. 6 B). When cells were grown in an environment
containing 200 mM or 800 mM CKI-7 neurite outgrowth of RGCs
was decreased by 98% and 100%, respectively. However, the
latter concentrations also significantly affected the survival of
RGCs (Fig. 6 B). Moreover, IC261 also significantly compromised
neurite outgrowth in a concentration dependent manner (Fig. 6
C). At concentrations of 0.5 mM and 1.5 mM, IC261 significantly
Figure 2. Localization of CK1d and e expression in RGCs. (A) Immunofluorescence staining and phase contrast image of the neurite growth
cone of a RGC using the CK1d specific monoclonal antibody 128A (red) and a bIII-tubulin specific monoclonal antibody (RB-9249-P0; green).
Epifluorescence microscopy of RGCs revealed that CK1d is located in granular particles aligned at microtubules all over the growth cone. Distribution
of CK1d in the soma is shown in the small vertical image panel (d: CK1d, tub: bIII-tubulin, m: merged). Scale bars: 10 mm. (B) Co-staining of CK1e
(serum 712; red) and bIII-tubulin (TUJ-1; green) in RGCs revealed a similar expression pattern as shown for CK1d in (A). Distribution of CK1e in the
soma is shown in the small vertical image panel (e: CK1e, tub: bIII-tubulin, m: merged). Scale bars: 10 mm.
Figure 3. Expression levels and localization of CK1d and e in PC12 cells. (A) Expression of CK1d and e in PC12 cells. CK1d and e expression
levels were detected in protein lysates of untreated and PC12 cells exposed to NGF for 1–48 h by western blot analyses using CK1d (128A) and CK1e
(#610446) specific antibodies. b-actin (detected on stripped membranes) served as loading control. (B) Densitometric evaluation of western blot
results shown in (A). Effects of NGF treatment are significant at * p,0.05 and ** p,0.01 when compared to untreated control cells. (C, D) Detection
of CK1d expression in PC12 cells after exposure to NGF for 48 h using antibodies for CK1d (serum NC10; red) and bIII-tubulin (MAB1637; green). CK1d
positive staining was observed in cell bodies, in neurites and in growth cones (C). Neurite and growth cone are presented at higher magnification in
(D). Scale bar for C: 20 mm. Scale bar for D: 2.5 mm. (E, F) CK1e immunostaining in PC12 cells after exposure to NGF for 48 h using antibodies for CK1e
(serum 712; red) and bIII-tubulin (MAB1637; green). CK1e was detected in the cytoplasm, in neurites and in the growth cones. A magnification of a
neurite and growth cone is presented in (F). Scale bar for E: 50 mm. Scale bar for F: 2 mm.
CK1d/e Activity Is Required for Neurite Outgrowth
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reduced neurite outgrowth by 10% and 30%, respectively. Neurite
outgrowth was completely abolished in the presence of 50 mM
IC261. However, IC261 did not affect the survival of RGCs in
culture at the concentrations tested (Fig. 6 C).
CK1-specific inhibitors destabilize neurite growth cones
Since the CK1-specific inhibitors CKI-7 and IC261 compro-
mised neurite outgrowth further experiments aimed to investigate
whether this effect was due to an inhibition of growth cone
initiation or to a destabilization of existing growth cones. For this
purpose retinal cultures were prepared from animals treated with
ONC+LI. After 24 h, when RGCs had already extended neurites,
control cells were fixed and others were exposed to vehicle or to
increasing concentrations of the CK1-specific inhibitors CKI-7 or
IC261. Cultures were incubated for another 24 h prior to fixation.
In all tested groups CK1-specific inhibitors did not significantly
affect the survival of RGCs (Fig. 7 B). The average neurite length
of untreated RGCs averaged 10.9 mm after 24 h and 27.1 mm
Figure 4. Kinase activities detected in PC12 cells after NGF treatment and in rat retina following ONC+ +LI. (A) Kinase activity in PC12
cells. Equal amounts of protein lysates from PC12 cells either untreated (control) or exposed to NGF for 24 h were loaded onto a 1 ml Resource-Q
column for ion exchange chromatography and eluted with a linear gradient of increasing NaCl concentration (represented as a solid diagonal line).
Fractions were collected and the CK1d/e specific kinase activity was determined in each single fraction as described in detail in Material and Methods.
Kinase activities (CPM: counts per minute) are represented by open squares (control) or closed triangles (24 h after NGF stimulation). (B) The kinase
activity in peak fraction 21 of untreated (control) and NGF stimulated PC12 cells (24 h) was significantly reduced by IC261 specifically inhibiting CK1d
and e in the micromolar range. Phosphate incorporation into GST-p531–64(FP267) was quantified by Cherenkov counting. (C) Kinase activity in rat
retina. Equal amounts of protein lysates from rat retinal tissue either untreated or treated with ONC+LI were loaded onto a 1 ml Resource-Q column
for ion exchange chromatography and eluted with a linear gradient of increasing NaCl concentration (represented as a solid diagonal line). Fractions
were collected and the CK1d/e specific kinase activity was determined in each single fraction as described in detail in Material and Methods. Kinase
activities (CPM: counts per minute) are represented by open squares (control) or closed triangles (ONC+LI). (D) The kinase activity in peak fraction 38
of extracts prepared from ONC+LI treated animals was significantly reduced by the CK1d/e specific inhibitor IC261. Phosphate incorporation into GST-
p531–64(FP267) was quantified by Cherenkov counting.
CK1d/e Activity Is Required for Neurite Outgrowth
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after 48 h in culture. Cultures treated with CKI-7 or IC261
showed significantly shorter neurites after 48 h than untreated
controls after 24 h (Fig. 7 A). These results suggest an essential role
for CK1 activity not only for neurite extension, but also for
maintaining the stability of the neurite and growth cone,
respectively. In order to confirm this hypothesis individual RGCs
with a highly dynamic neurite growth cone were recorded by time-
lapse microscopy in the absence and presence of either IC261 or
D4476, another CK1-specific inhibitor, which blocks CK1d and e
activity already in the low micromolar range . IC261 (50 mM)
induced growth cone collapse and neurite retraction of RGCs
(Fig. 7 C). Neurite length was reduced by 50% within 70 min and
almost totally retracted after 3 h (Fig. 7 C, D). The cell body of the
RGC remained unaffected. Similar results were obtained in the
presence of D4476, which induced growth cone collapse and
neurite retraction of RGCs at 5 mM (Fig. 7 C, D and video
sequence in supporting data file S2). Neurite retraction was almost
complete after 4 h, whereas the cell body of the RGC remained
unaffected. Control cells were treated with vehicle only (DMSO,
transfection reagent; Fig. 7 C, D and video sequence in supporting
data file S1).
The main findings of the current study are: (1) CK1d and CK1e
are localized in neurites and growth cones of PC12 cells and
regenerating RGCs, (2) CK1d/e activity is increased in differen-
tiating PC12 cells and regenerating retina and (3) CK1-specific
small molecule inhibitors destabilize existing growth cones and
compromise neurite growth of RGCs and PC12 cells. These
observations suggest that the activity of CK1d and e is essentially
involved and necessary for neurite outgrowth and regeneration.
Cellular kinases and phosphatases are involved in complex
signaling during neuronal degeneration and regeneration leading
to remodeling of the cytoskeletal architecture [60–63]. Members of
the CK1 family reportedly play an important role in cytoskeletal
rearrangements (reviewed in ) by mediating hyperphosphor-
ylation of the microtubule-associated protein tau, which is
associated with Alzheimer’s disease [48,64–69]. Despite the role
of CK1 family members in neurodegenerative disorders, an
involvement of these kinases in neurite growth or axonal
regeneration has not yet been reported.
Here, we first analyzed the localization of CK1d and e in
primary, mature RGCs and PC12 cells. Both CK1 isoforms were
located in the soma and were aligned in granular structures along
microtubules. In addition, we found both isoforms located in the
growth cones of regenerating RGCs and PC12 cells. Second we
analyzed the occurrence of changes in the expression and activity
of CK1d and e in regenerating RGCs and differentiating PC12
cells. No notable changes in the expression levels of CK1d and e
were detected in retinas after optic nerve injury or when RGCs
entered into a regenerative state after additional LI. NGF
stimulated PC12 cells showed only a slight and temporary
upregulation of CK1d expression and a transient decrease in the
Figure 5. Characterization of CK1d C-terminal targeting cellular kinase activities. Equal protein amounts of lysates from untreated PC12
cells or PC12 cells exposed to NGF for 24 h were loaded onto a 1 ml Resource-Q column for ion exchange chromatography and eluted with a linear
gradient of increasing NaCl concentration (represented as a solid diagonal line). Kinase activity was determined in each single fraction using the GST-
CK1d305–375fusion protein (FP1006) as substrate. Quantification of phosphate incorporation was measured by Cherenkov counting. The major kinase
activity eluted at 340–410 mM NaCl in extracts prepared before (open squares) or 24 h (closed triangles) after the induction of differentiation
processes of PC12 cells. Data in Fig. 5 show one representative experiment of two.
CK1d/e Activity Is Required for Neurite Outgrowth
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Figure 6. CK1d and e specific inhibitors compromise neurite outgrowth of RGCs and PC12 cells. (A) PC12 cells were cultured for 3 days
without NGF (control) and in the presence of NGF+vehicle or NGF+CKI-7 (50 and 200 mM) or IC261 (0.5, 1.5 and 50 mM). In a concentration dependent
manner the average neurite length of NGF stimulated PC12 cells was markedly reduced in the presence of the CK1-specific inhibitors CKI-7 or IC261.
Treatment effects: **p,0.01 and ***p,0.001 compared to cells exposed to NGF+vehicle. (B, C) LI stimulated RGCs were cultured in the absence
(control) or presence of CKI-7 (B; 50, 200 and 800 mM) or IC261 (C; 0.5, 1.5 and 50 mM). Neurite outgrowth was compromised in a concentration
dependent manner. The survival of RGCs was only affected in the presence of CKI-7 at 800 mM. Treatment effects compared to control groups:
*p,0.05, **p,0.01, ***p,0.001.
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Figure 7. Inhibition of CK1d and e destabilizes neurites and growth cones of regenerating RGCs. (A) LI stimulated RGCs were cultured for
24 h and then exposed to vehicle or different concentrations of the CK1-specific inhibitors IC261 (0.5, 1.5 and 50 mM) or CKI-7 (50 and 200 mM). The
average neurite length of RGCs was determined after 24 h (first control) and 48 h, respectively. Treatment effects: ***p,0.001. (B) The number of
RGCs per well in all groups was not affected by any treatment described in (A). (C) LI stimulated RGCs were cultured for 48 h and subsequently
monitored in a flow-through chamber for live cell imaging. In the presence of vehicle or inhibitors IC261 (50 mM) or D4476 (5 mM) cells were recorded
for 4 h. Retraction of the outgrown neurite after exposure to IC261 and D4476 is shown at 0, 120 and 240 min. Arrowheads indicate the position of
the cell body, arrows the position of the growth cone. Scale bar: 50 mm. (D) Quantification of neurite retraction of RGCs over time exposed to vehicle
(control, closed squares) or inhibitors IC261 (50 mM, open triangles) or D4476 (5 mM, open squares) specifically inhibiting CK1d and e.
CK1d/e Activity Is Required for Neurite Outgrowth
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expression of CK1e. However, a 3-fold increase in the CK1d/e
specific kinase activity was observed in differentiating PC12 cells
compared with untreated controls. The CK1d/e specific kinase
activity was confirmed by its reduction in the presence of small
molecule inhibitors specific for CK1d and e. These data suggest
that the increased CK1d/e activity is not basically regulated by the
expression levels of CK1d and e, but rather through an alternative
mechanism. Similar findings were also made in retinal tissue
derived from untreated rats and animals that were subjected to
ONC+LI. However, CK1d/e kinase activities detected in the
current study were eluted at a higher NaCl concentration most
likely due to changes in the phosphorylation state of CK1d and e
proteins. Such changes in the chromatographic properties of
CK1d and e after an isolation from various tissues and cell lines
are consistent with previous reports [70,71]. The activity of CK1d
is reportedly modulated by several cellular kinases, such as PKA,
specifically phosphorylating CK1d within its C-terminal domain
. Coherently, we found a reduction in the activity of cellular
CK1d C-terminal targeting kinases in NGF differentiated PC12
cells, suggesting that the increased activity of CK1d may have
been mediated indirectly through altered activity of CK1d C-
terminal targeting kinases. However, additional experiments are
necessary to identify this (these) cellular kinase(s) and to
characterize their physiological interactions with CK1d and their
role in regulating neurite outgrowth.
Finally, in the current study we demonstrate that pharmacolog-
ical inhibition of CK1d/e activity by two different ATP-competitive
small molecule inhibitors markedly compromised neurite outgrowth
and induced a destabilization of neurite growth cones of RGCs and
PC12 cells. CKI-7 and IC261 effectively blocked neurite outgrowth
of regenerating RGCs and PC12 cells at concentrations that did not
affect the survival of these cells. Moreover, the fact that all tested
CK1d/e specific inhibitors, namely IC261, CKI-7 and D4476,
exhibited similar results on neurite growth minimizes the possibility
of unspecific effects . Thus, the results of the pharmacological
inhibition of CK1 imply that CK1d/e activity is necessary for
neurite outgrowth of primary RGCs. This assumption was further
underlined by time-lapse analyses showing that the CK1d and e
specific inhibitors IC261 and D4476 induced a collapse and
retraction of growing neurites of isolated RGCs, probably due to a
destabilization of the microtubule and/or actin cable network. This
hypothesis is supported by previous observations showing that CK1
is involved in regulating both microtubule and actin filament
In orderto obtainmore information regardingthepossibleroleof
CK1 family members in maintaining microtuble integrity their
functions in modulating the interaction of a/b-tubulin with
microtubule associated proteins (MAPs) should be analyzed more
in detail in the future. Additional experiments are also required to
clarify theroleof CK1d and e inregulatingactin filament dynamics.
Although the current study suggests that CK1d/e activity is
required for neurite outgrowth further studies need to be performed
to test the possibility as to whether enhancing the activity of CK1d
and e may facilitate neurite outgrowth and be useful for the
development of therapeutic concepts to stimulate axonal regener-
ation. These experiments may become possible when specific
activators either for CK1d or e will be available in the future.
Materials and Methods
Optic nerve cut and lens injury
All animals were housed and handled in accordance to official
regulations for care and use of laboratory animals and maintained
under SPF conditions. Ethical approval of all experiments and
surgical procedures was approved by the local authorities
(Regierungspra ¨sidium Tu ¨bingen, permission number 1011).
Adult female Sprague-Dawley rats weighing 220–250 g were
anesthetized by intraperitoneal injections of ketamine (60–80 mg/
kg) and xylazine (10–15 mg/kg), and a 1–1.5 cm incision was
made in the skin above the right orbit. The optic nerve was
surgically exposed under an operating microscope, the dural
sheath was longitudinally opened and the nerve was cut 1 mm
behind the eye, avoiding injury to the central retinal artery. The
vascular integrity of the retina was verified by fundoscopic
examination. Lens injury (LI) was induced by a retrolenticular
approach, puncturing the lens capsule with the tip of a
microcapillary tube as described previously [7,9].
Isolation of RGCs and tissue culture
Five days after surgical treatment animals were killed by an
overdose of chloralhydrate solution (14%) and dissected retinal tissue
was incubated in Dulbecco’s Modified Eagle medium (DMEM)
(Invitrogen, Karlsruhe, Germany) containing papain (16.4 U/ml,
Worthington, Katarinen, Germany) and L-cysteine (0.3 mg/ml,
Sigma-Aldrich, Munich, Germany) for 30 min at 37uC as described
previously . After digestion, the retinal tissue was rinsed twice
with DMEM before being transferred into DMEM containing B27-
supplement (1:50, Gibco, Karlsruhe, Germany) and penicillin/
streptomycin (0.2 mg/ml, Biochrom, Berlin, Germany). Then, the
triturated retina was passed through a cell strainer before seeding of
the isolated RGCs onto poly-D-lysine (0.1 mg/ml, molecular weight
,300000 Da, Sigma-Aldrich, Munich, Germany) and laminin
(20 mg/ml, Sigma-Aldrich, Munich, Germany) coated culture dishes.
PC12 cells  were seeded on poly-D-lysine coated tissue
culture dishes and maintained in DMEM supplemented with 10%
horse serum, 5% fetal calf serum (both Gibco, Karlsruhe,
Germany) and penicillin/streptomycin (0.2 mg/ml, Biochrom,
Berlin, Germany) at 37uC in a humidified atmosphere (85%
humidity) containing 5% CO2. In neurite outgrowth assays, cells
were treated with NGF (100 ng/ml) as indicated, the CK1-specific
kinase inhibitors CKI-7 (Sigma-Aldrich, Munich, Germany) 
and IC261 (ICOS Corporation, Bothell, USA)  were added
directly after seeding. In each case neurite outgrowth was
quantified using the public domain image processing software
ImageJ (National Institutes of Health, Bethesda, USA). The
significances of intergroup differences were evaluated using a
one-way analysis of variance (ANOVA) test, followed by a
correction of a post hoc test (Turkey). All data are provided as
average and standard error (SEM).
Time-lapse microscopy of RGCs
For live cell imaging, RGCs were grown on glass slides for 48 h
and then transferred to a flow-through chamber for inverted
microscopes (Bioptechs, Butler, USA) and further cultivated in
supplemented RPMI (Invitrogen, Karlsruhe, Germany; containing
B27, 1:50) at a medium flow-rate of 1 ml/h. After 30 min,
medium was exchanged to growth medium containing the CK1-
specific inhibitors IC261 (ICOS Corporation, Bothell, USA) 
or D4476 (Calbiochem, Darmstadt, Germany)  or DMSO/
transfection reagent (as a negative control). D4476 was applied
using transfection reagent (Effectene, Qiagen, Hilden, Germany)
as described previously . The cells were monitored for 4 h and
phase contrast or bright field time-lapse recordings were taken
under 406 magnification using the Olympus IX81 microscope
(Olympus, Hamburg, Germany) and the CellRsoftware.
Neurite length was quantified every 10 min before and during
cell treatment using the public domain image processing software
ImageJ (National Institutes of Health, Bethesda, USA).
CK1d/e Activity Is Required for Neurite Outgrowth
PLoS ONE | www.plosone.org8June 2011 | Volume 6 | Issue 6 | e20857
For immunofluorescence staining monoclonal antibodies spe-
cific for bIII-tubulin were purchased from Babco (Richmond,
MAB1637, mouse, 1:1000) and Thermo Fisher Scientific (Fre-
mont, USA; RB-9249-P0; rabbit; 1:500). For detection of CK1d
the mouse monoclonal antibody 128A (ICOS Corporation,
Bothell, USA; 1:500) and the rabbit polyclonal serum NC10
 (1:200) were used. CK1e was detected using rabbit polyclonal
serum 712  (1:200). Fluorescence labeled secondary antibodies
anti-mouse/anti-rabbit Alexa Fluor 488 or anti-rabbit/anti-mouse
Alexa Fluor 633 (each 1:1000) were supplied by Molecular Probes
In western blot analyses, mouse monoclonal antibodies against
CK1d (128A, ICOS Corporation, Bothell, USA; 1:5000), CK1e
(#610446, Becton Dickinson, Franklin Lakes, USA; 1:150), or b-
actin (A5441, Sigma-Aldrich, Munich, Germany; 1:10000) were
used. Immunocomplexes were detected using anti-mouse HRP
conjugated IgG (both 1:10000, GE Healthcare, Chalfont St Giles,
Millipore (Billerica, USA;
Cells grown on coated coverslips were washed twice with PBS
and fixed in 4% paraformaldehyde for 30 min. Fixed cells were
permeabilized using 0.3% Triton X-100 and blocked with 2%
BSA in PBS/Tween (0.05%) for 1 h followed by incubation with
primary antibodies for 45 min at room temperature or over night
at 4uC. After washing with PBS secondary fluorescence labeled
secondary antibodies were applied for 30 min at room tempera-
ture. Finally, cells were embedded in mounting medium
containing 5% polyvinyl alcohol (MW 70000–100000 Da, Sig-
ma-Aldrich, Munich, Germany) and 10% glycerol (Roth,
Karlsruhe, Germany) in PBS. Analyses and documentation were
done using a fluorescence microscope and a high-resolution digital
camera (Olympus, Hamburg, Germany).
Overexpression and purification of glutathione S-
transferase fusion proteins
The production and purification of the GST-fusion proteins
FP267 (GST-p531–64) and FP1006 (GST-CK1d305–375) were
carried out as described elsewhere [55,57].
Fractionation of proteins
Untreated and differentiated PC12 cells were washed with PBS
and lysed in sucrose lysis buffer containing 20 mM Tris-HCl
[pH 7.0], 0.27 mM sucrose, 1% Triton X-100, 1 mM EGTA,
1 mM benzamidine, 50 mM leupeptin, 1% Trasylol (aprotinin),
and 0.1% b-mercaptoethanol (MSH) on ice. Cell lysates were
passed through a 0.45 mm filter and in each case 25 mg of total
protein were applied to an anion exchange column (Resource-Q
1 ml) attached to an ETTAN LC purifier (both GE Healthcare,
Chalfont St Giles, GB). Proteins were eluted with a linear
ascending gradient between 0–1000 mM NaCl in 50 mM Tris-
HCl [pH 7.5], 1 mM EDTA, 5% glycerol, 0.04% Brij, 1 mM
benzamidine, 4 mg/ml leupeptin, 1% Trasylol (aprotinin) and
0.1% b-mercaptoethanol. Fractions of 250 ml volume were
collected. Aliquots of 2 ml of each fraction were used for in vitro
kinase assays to determine kinase activities in single fractions.
In vitro kinase assays
In vitro kinase assays were carried out in the absence or presence
of the CK1-specific inhibitor IC261  in kinase buffer
containing 100 nM ATP, 25 mM Tris-HCl [pH 7.5], 10 mM
MgCl2, 0.1 mM EDTA and 2 mCi [32P] c-ATP. GST0p531–64
(FP267, [55,57]) and GST-CK1d305–375(FP1006, ) were used
as substrates. Single fractions of fractionated PC12 cell extracts
were used as sources of enzyme. Phosphorylated proteins were
separated by SDS-PAGE and the protein bands were visualized on
dried gels by autoradiography. Phosphorylated protein bands were
excised and quantified by Cherenkov counting.
Western blot analysis
For the detection of CK1d and CK1e in retinal tissue of
untreated and treated animals and differentiated PC12 cells,
respectively, protein lysates were prepared in lysis buffer
containing 50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 0.5%
NP40, 10% glycerol, 1 mM EGTA, 1 mM benzamidine, 50 mM
leupeptin, 1% Trasylol (aprotinin) and 5 mM DTT. Extracts were
clarified by centrifugation. 75 mg of each protein extract were
separated by SDS-PAGE and transferred onto a PVDF blotting
membrane (Hybond P, GE Healthcare, Chalfont St Giles, GB).
The membranes were probed with primary antibodies and
immunocomplexes were detected using HRP conjugated IgG
followed by chemiluminescence detection. Where indicated
membranes were stripped before being used for reblotting.
Supporting data file S1
treated with DMSO/transfection reagent. After 48 h in
culture, dissociated LI stimulated RGCs were monitored in a flow-
through chamber for live cell imaging. As indicated, cells were
treated with vehicle (DMSO and transfection reagent). Time-lapse
recordings were taken for 4 h.
Time-lapse recording of RGCs
Supporting data file S2
treated with 5 mM D4476. After 48 h in culture, dissociated LI
stimulated RGCs were monitored in a flow-through chamber for
live cell imaging. As indicated, cells were treated with the inhibitor
D4476 (5 mM) specifically inhibiting CK1d and e, using transfection
reagent (Effectene, Qiagen, Hilden, Germany) as described
previously . Time-lapse recordings were taken for 4 h.
Time-lapse recording of RGCs
We would like to thank Tony DeMaggio (ICOS Corporation, USA) for
providing us with the CK1d specific monoclonal antibody 128A, Anastasia
Andreadakis, Sabrina Winter, Annette Blatz and Bernhard Schmidt for
technical assistance, and Martin Sto ¨ter for assistance, suggestions and
Conceived and designed the experiments: UK DF. Performed the
experiments: JB AM MF. Analyzed the data: JB AM MF DF UK.
Contributed reagents/materials/analysis tools: UK DF JB AM MF. Wrote
the paper: JB DF UK.
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