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JOURNAL OF NEUROCHEMISTRY
| 2014 | 130 | 678–692
, ,1
doi: 10.1111/jnc.12740
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*Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University
School of Medicine, Shanghai, China
†University of Chinese Academy of Sciences, Beijing, China
‡Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese
Ministry of Education, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
§Shanghai Stem Cell Institute, Institute of Medical Sciences, Shanghai JiaoTong University School of
Medicine, Shanghai, China
Abstract
For our nervous system to function properly, each neuron
must generate a single axon and elongate the axon to reach
its target. It is known that actin filaments and their dynamic
interaction with microtubules within growth cones play important roles in inducing axon extension. However, it remains
unclear how cytoskeletal dynamics is controlled in growth
cones. In this study, we report that Rufy3, a RUN domaincontaining protein, is a neuron-specific and actin filamentrelevant protein. We find that the appropriate expression of
Rufy3 in mouse hippocampal neurons is required for the
development of a single axon and axon growth. Our results
show that Rufy3 specifically interacts with actin filamentbinding proteins, such as Fascin, and colocalizes with
Fascin in growth cones. Knockdown of Rufy3 impairs the
distribution of Fascin and actin filaments, accompanied by an
increased proportion of neurons with multiple axons and a
decrease in the axon length. Therefore, Rufy3 may be
particularly important for neuronal axon elongation by interacting with Fascin to control actin filament organization in
axonal growth cones.
Keywords: axon length, F-actin, Fascin, growth cones,
Rufy3.
J. Neurochem. (2014) 130, 678–692.
Cover Image for this issue: doi: 10.1111/jnc.12580.
A properly connected and functional nervous system is
dependent on the ability of neurons to develop polarity
through forming a single axon and multiple dendrites.
Knowledge of how the axon forms and elongates is critical
for understanding the development of the nervous system.
Cultured neurons from the mammalian hippocampus have
been widely used to study the regulation of neuronal axon
development (Craig and Banker 1994). In culture, these
neurons form several morphologically similar neurites before
polarization occurs. One of the neurites then grows significantly faster than others and becomes an axon, whereas
others become dendrites (Dotti and Banker 1987; Goslin and
Banker 1989). At the tip of a growing axon, there is a highly
dynamic, sensory-motile structure, specialized for elongation
and steering, known as a growth cone. It is known that
neuronal growth cones play critical roles in guiding axons to
678
Received November 22, 2013; revised manuscript received April 2,
2014; accepted April 7, 2014.
Address correspondence and reprint requests to Ying Jin, Institute of
Health Sciences, 225 South Chongqing Road, Shanghai, 200025, China.
E-mail: [email protected]
1
These authors contributed equally to this work.
Abbreviations used: bFGF, basic fibroblast growth factor; BME,
b-mercaptoethanol; BSA, bovine serum albumin; DIC, differential
interference contrast microscope; DMSO, dimethyl sulfoxide; EGF,
epidermal growth factor; EGFP, enhanced green fluorescent protein;
ESCs, embryonic stem cells; FBS, fetal bovine serum; GST, glutathioneS-transferase; IB, immunoblotting; IP, immunoprecipitation; IRES,
internal ribosome entry sites; LIF, leukemia inhibitory factor; Map2,
microtubule associated protein 2; MS, mass spectrometry; NEAA, nonessential amino acid; NP-40, nonyl phenoxypolyethoxylethanol; NPCs,
neural progenitor cells; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RNAi, RNA interference; Rufy3 OE, Rufy3 overexpression; SDS–PAGE, dodecyl sulfatesodium salt–polyacrylamide gel
electrophoresis.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
Rufy3 controls axon growth
their appropriate targets. Operationally, the growth cone has
been divided into three primary domains. The central
domain, located at the end of a growing axon, is the most
proximal part of the growth cone and is filled with abundant
microtubules and vesicles. The peripheral domain is at the
most distal area of the growth cone and is motile; it is
composed of bundles of actin filaments (F-actin) and fringed
with lamellipodia and filopodia. The transitional domain is
located at the junction between the central and peripheral
domains. It contains overlapping networks of F-actin and
microtubules (Lin and Forscher 1993; Geraldo and GordonWeeks 2009). A previous study demonstrated that local
instability of the actin network in a single growth cone
determines whether it becomes an axon or a dendrite,
suggesting that the higher rate of actin turnover may underlie
neuronal polarization (Bradke and Dotti 1999). Therefore,
considerable attention in recent years has been centered on
understanding how actin cytoskeleton and its interaction with
microtubules are precisely modulated in the growth cone.
There are two kinds of cytoskeletal filaments, F-actin and
microtubules, in the growth cone. The actin cytoskeleton,
composed of actin polymers and a large variety of associated
proteins, is a highly dynamic network (Schmidt and Hall
1998). Actin bundles distributed throughout the lamellipodium extend beyond the leading edge of the growth cone to
form the core of filopodia (Bentley and Toroian-Raymond
1986). Although how exactly filopodia form is not entirely
clear, some actin-associated proteins have been found in
growth cones and are known to play an important role in the
regulation of growth cone morphology and behavior. For
instance, Fascin, a conserved actin-bundling protein, has
been reported to play a role in defining the dendrite
morphology in neurons of Drosophila larvae (Nagel et al.
2012), forming filopodia of tumor cells (Sun et al. 2011) as
well as forming and maintaining actin bundles in Helisoma
neuronal growth cone (Cohan et al. 2001). Moreover, the Factin side-binding protein, Drebrin, has been shown to
localize at the transitional domain of growth cones and is
involved in dendritic (Hayashi and Shirao 1999; Mizui et al.
2005) and axonal development (Mizui et al. 2009). Furthermore, Drebrin was reported to inhibit the bundling activity of
Fascin. Exploring how these F-actin-binding proteins participate in axon growth and identifying new actin-associated
factors are important for our understanding of the molecular
mechanism underlying the formation and elongation of
neuronal growth cones.
In this study, we characterize the role of Rufy3, a RUN
domain-containing protein, in the morphogenesis of neural
axons and explore the potential molecular basis underlying its
function in primary mouse hippocampal neuron culture.
Rufy3 was chosen as the focus of this study because of the
reported role of its rat ortholog, named Singar 1, for
suppressing surplus axon formation in a PI3K activitydependent manner in rat embryonic hippocampal neurons
679
(Mori et al. 2007). Nevertheless, it remains unknown whether
Rufy3 would exhibit additional roles in neurons besides
suppressing surplus axons, and if so, how the function is
executed. Here, we report that Rufy3 is exclusively expressed
in the neurons of the mouse developing brain. Knockdown of
Rufy3 leads to shortened axons in addition to an increased
fraction of neurons with multiple axons. Conversely, Rufy3
over-expression results in an increase in the axon length.
Moreover, we find that Rufy3 interacts with Fascin and
Drebrin, codistributing with F-actin in the axonal growth
cone. Silencing of Rufy3 disrupts the normal distribution of
Fascin and F-actin. The findings are significant for understanding how neuronal axon formation and growth cone
morphogenesis are controlled at a molecular level.
Materials and methods
Animal, cell culture, and drug treatment
Mice of the C57/BL6 strain used in this study were purchased from
SLAC, Shanghai, China. All animal procedures were performed
according to the guidelines approved by the Shanghai JiaoTong
University School of Medicine (20080050) and conformed to the
NIH guideline for the use of animals in research. Every effort was
made to minimize animal suffering and reduce the number of
animals used.
HEK293T cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) (Gibco/Life Technologies, Grand Island, NY,
USA) supplemented with 10% fetal bovine serum (Hyclone/ GE
Healthcare, Piscataway, NJ, USA), 2 mM L-glutamine, 1 unit/mL
penicillin, and 100 lg/mL streptomycin, and passaged every
2 days. After transfection with plasmids for 48 h, cell lysates were
collected for coimmunoprecipitation (Co-IP) experiments. Mouse
hippocampal and cortical neurons prepared from post-natal day 0
(P0) C57/BL6 mouse embryos were seeded on coverslips coated
with poly-D-lysine (Millipore, Temecula, CA, USA) and laminin
(Invitrogen/Life Technologies, Grand Island, NY, USA), and
cultured in Neurobasal medium (Gibco) supplemented with B27
supplement (Invitrogen), 2 mM L-glutamine (Gibco) and 1% nonessential amino acid (NEAA, Gibco), without a glial feeder layer, as
described previously (Ha et al. 2009). The D3 mouse embryonic
stem cells (ESCs) were maintained in DMEM supplemented with
10% fetal bovine serum (Hyclone), 1% NEAA, 100 lM bMercaptoethanol (Gibco), 2 mM L-glutamine, 1 unit/mL penicillin,
100 lg/mL streptomycin, and 1000 units/mL leukemia inhibitor
factor (LIF, Millipore) on mouse embryonic fibroblast (MEF), and
passaged using 0.05% trypsin every 3 days, as described previously
(Tremml et al. 2008). For cytochalasin D (CytoD, Sigma, St Louis,
MO, USA) treatment, hippocampal neurons cultured for 48 h were
incubated with 1 lM of CytoD for 2 h before fixation.
DNA constructs
Full-length cDNA of the mouse Rufy3 was amplified by PCR using
cDNA from a mouse E10.5 brain as a template. The cDNA was then
subcloned into the pCAG-IRES-EGFP plasmid described previously
(Lorsbach et al. 2004). For the study of protein interaction, the
cDNA was transferred into a Flag-pPy vector as described
previously (Li et al. 2009). Full-length cDNA of mouse Drebrin
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
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was amplified by PCR using cDNAs from a mouse E18.5 brain as a
template and the longest transcript was subcloned into the HA-pPy
vector. Full-length cDNA of mouse Fascin, the N terminus of Rufy3,
and the C terminus of Rufy3 were amplified in the same way as for
Drebrin. For RNA interference, two shRNA duplexes (Thermo
Scientific, Wilmington, DE, USA) against Rufy3, shRufy3-1: 50 - AGA
AATGGAACGAGTTAAA -30 and shRufy3-2: 50 - TAAACAA
CCTTCAGACAAA -30 , were, respectively, cloned into a pLKO.3G
vector purchased from the Addgene (named Ri-1# and Ri-2#,
respectively), and the empty pLKO.3G vector was used as a control.
The sequences of all constructs were verified by DNA sequencing.
Transfection and coimmunoprecipitation
Plasmid transfection was performed using Lipofectamine 2000
(Invitrogen). For Co-IP experiments with exogenously expressed
proteins, cell extracts were collected 48 h after transfection and
immunoprecipitated by anti-Flag antibody, as described previously
(Li et al. 2007). For Co-IP experiments with endogenous proteins,
whole-brain extracts from C57/BL6 P0 mice were extracted in CoIP buffer containing 50 mM Tris-HCl pH 7.5 (Sangon, Shanghai,
China), 150 mM NaCl (Sangon), 2 mM EDTA, pH 8.0 (Sangon),
10% glycerol (Sangon) and, 0.5% nonyl phenoxypolyethoxylethanol (NP-40, Sangon), then immunoprecipitated by antibodies
against Rufy3, Drebrin or Fascin, respectively.
Mouse ESC neural differentiation
Neural differentiation from D3 mouse ESCs was induced as
previously described (Watanabe et al. 2005). ESCs were cultured
in suspension in a serum-free medium for 7 days for the formation
of embryoid bodies (EBs). The EBs were then plated onto dishes
coated with Matrigel and passaged every 3 days. After differentiation for 7–10 days, many NPCs and a few neurons appeared.
Subsequently, the number of neurons increased dramatically with
the concomitant decrease in the number of NPCs. At 19 days of
differentiation, the number of neurons decreased with an increase in
the number of glia cells.
Primary neuron differentiation
The brain cortex of the C57/BL6 E14.5 mouse was dissected and
digested. The dissociated cells were suspended as neurospheres in
the DMEM/F12 medium (Gibco) supplemented with N2 (Invitrogen) and Neurobasal medium plus B27 (N2B27 medium) with
bFGF (R&D, 10 ng/mL) and EGF (R&D, 10 ng/mL) for 3 day as
described previously (Wu et al. 2001). On the fourth day, the
spheres were digested and plated onto Matrigel-coated (BD) dishes
in the N2B27 medium without bFGF and EGF for 6 days. Samples
were collected at day 0 (the day 3 – neurosphere stage), day 1 (the
first day when the cells were plated onto dishes), day 2, and day 6.
Antibodies
To generate the antibody that specifically recognizes Rufy3, we
expressed a GST-fused Rufy3 fragment (residues 567-807) and
purified the fusion proteins from E. coli BL21 (DE3) to immunize
rabbits. Anti-serum was affinity purified as previously reported (Xu
et al. 2004). The following antibodies were purchased from commercial sources: a-Tublin from Sigma, Nestin from Millipore, Map2 from
Millipore, Tuj1 from Invitrogen, Drebrin from Thermo, Fascin from
Epitomics Inc, SMI312 from Abcam (Cambridge, MA, USA),
Gapdh from Boster (Wuhan, Hubei, China), enhanced green
fluorescent protein (EGFP) from Roche (Indianapolis, IN, USA)
and Flag from Sigma.
Immunofluorescence staining, western blot, and microscopic
analyses
Cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde (Sangon) for 10 min, permeabilized with a solution containing
0.5% Triton X-100 for 3 min, and blocked with 3% bovine serum
albumin for 30 min at 25°C. Then, the cells were incubated with
antibodies against Rufy3 (1 : 500 dilution), Fascin (1 : 200
dilution), Drebrin (1 : 1000 dilution) or EGFP (1 :500 dilution),
respectively. After extensive washing with phosphate-buffered
saline for three times, the cells were incubated with FITCconjugated or CY3-conjugated secondary antibodies (Proteintech,
Chicago, IL, USA) at 37°C for 1 h. F-actin was labeled with
phalloidin from Invitrogen and the nuclei were counter-stained with
4, 6-diamidino-2 phenylindole. Finally, images of cells were
captured (TCS SP5; Leica Microsystems, Wetzlar, Germany). Brain
sections were stained as the cells were, but longer fixation times and
antibody incubation times were used. Western blot analyses were
conducted as described previously (Liao and Jin 2010).
Mass spectrometry
To identify proteins interacting with Rufy3, whole-brain proteins
from C57/BL6 P0 mice were extracted using the Co-IP buffer.
About 4 mg of brain extract was incubated with Rufy3 antibodies
overnight. On the next day, the mixture was incubated with protein
A beads for 3 h at 4°C to precipitate Rufy3 and associated proteins.
IgG was used as a negative control for the Rufy3 antibody.
Endogenous Rufy3 and proteins forming protein complexes with
Rufy3 in brain cells were separated by SDS–PAGE and visualized
by Coomassie Brilliant Blue staining. The gels were cut into 7–10
pieces for both IgG- and Rufy3 antibody-precipitated samples.
Proteins in the gels were then sequenced by mass spectrometry in
BIDMC Proteomics Center and Dana Farber/Harvard Cancer
Center, Cancer Proteomics Core in Beth Israel Deaconess Medical
Center at Harvard Medical School. The mass spectrometry spectra
were acquired using the mass spec analysis program called Protein
Pilot, version 3.0 reported in a previous study (Shilov et al. 2007).
To find out relevant functions and pathways from the identified
protein list, the enrichment analysis was carried out using the
DAVID website. Firstly, various annotations were extracted automatically by the tool. Then annotation clustering analysis was
performed by choosing the three categories of Gene Ontology (GO),
including Biological Process (BP), Cellular Component (CC), and
Molecular Function (MF), which could reveal common functions
among the proteins. Finally, 18 KEGG pathways were mapped for
the protein list according to the previous study (da Huang et al.
2009).
Statistical analyses
The significance for the difference between two groups was
determined by an unpaired Student’s t-test. A neurite was considered as an axon when it was longer than 100 lm and its length was
twice that of other neurites. The axon length was measured by the
Image-Pro Plus. All experiments were conducted at least three times
independently. Data are shown as mean SD.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
Rufy3 controls axon growth
(a)
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(c)
(d)
(b)
(e)
Fig. 1 The expression pattern of Rufy3 in mouse brains. (a) Rufy3
antibody recognizes a single Rufy3 band in brain extracts. Whole
tissue extracts from different organs of post-natal day 0 (P0) mice
were prepared and immunoblotted with the affinity-purified Rufy3
polyclonal antibody. Rufy3 proteins were only detected in the brain.
(b) Protein levels of Rufy3 in the brain at different stages of mouse
development determined by western blot analysis. (c) The distribution of Rufy3 proteins in the brain of an E11.5 mouse detected by
immunofluorescence staining. The right panel is an enlarged image
of the inbox shown in the left panel. The Rufy3 antibody was used
to stain endogenous Rufy3 proteins (red). (d) The distribution of
Rufy3 in the brain of an E18.5 mouse was determined by double
staining with antibodies against Rufy3 (red) and Tuj1 (green) or
Nestin (green). Merged images with higher magnification are shown
in the right panels. (e) Immunofluorescence images to show the
distribution of Rufy3 in the cortex of an E18.5 mouse brain. The
brain sections were double stained with antibodies against Rufy3
(red) and Nestin (green), or Tuj1 (green) or Map2 (green). Thin
white lines are used to highlight the borderline of the cortical plate
(CP), intermediate zone (IZ), and ventricular zone (VZ) of the brain.
Merged images with higher magnification are shown in the right
panels. Scale bars: 500 lm in the left panel of (c); 200 lm in the
right panel of (c) 100 lm in (d); 250 lm in the left four panels of (e);
100 lm in the right panel of (e).
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
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Z. Wei et al.
Results
Rufy3 is detected in the neuron-rich regions of the brain
Our previous study found that RUFY3 had an expression
pattern similar to that of neuronal markers, such as TUJ1 and
MAP2, in the human embryo during human embryonic days
20–32, suggesting that Rufy3 might be expressed in the human
developing nervous system (Figure S1) (Fang et al. 2010). In
addition, its ortholog was detected in the rat brain (Mori et al.
2007). However, its temporal and spatial expression pattern in
the mouse remains largely unclear. To address this issue, we
generated an affinity-purified polyclonal antibody against
Rufy3. The antibody specifically recognized endogenous
Rufy3 proteins, but not its other family members, as it detected
only a single protein band when western blot analysis was
conducted using whole-cell lysates of various mouse organs
(Fig. 1a). Among four members of the mouse Rufy3 family,
we found that Rufy3 was only expressed in the brain, whereas
Rufy1 and Rufy2 were found in other mouse organs in addition
to the brain, and Rufy4 was not detected in the brain
(Figure S2a and b). At P0, mouse Rufy3 proteins were only
detected in the brain and not in the other organs tested,
including the heart, lung, liver, and kidney, verifying it as a
brain-specific protein (Fig. 1a). During embryonic development, Rufy3 expression was detected in the brain from
embryonic day 13.5 (E13.5) to adult, peaking around P0 and
decreasing gradually with development (Fig. 1b). To gain
more information of its spatial expression profile, embryonic
sections of different developmental stages were used in the
immunofluorescence staining. In sections of the whole embryo
at E11.5, we found expression of Rufy3 in the cortex of the
central nervous system, where it appeared restricted to the upper
layers, but was not found in the ventricular zone (VZ) (Fig. 1c).
Moreover, results from double staining of brain sections of
E18.5 embryos with antibodies against Rufy3 and Tuj1 (a
neuronal marker) or Nestin (a neural progenitor cell, NPC,
Fig. 2 Expression of Rufy3 in mouse primary neurons and during
mouse ESC neural differentiation. (a) The cell morphology of neuronal
differentiation from primary cortical neural progenitor cells (NPCs).
NPCs were isolated from E14.5 mouse brain cortex and suspended as
neurospheres in culture. On the third day, spheres were digested and
plated onto Matrigel-coated dishes and differentiated for 6 days.
Images of the bright field were taken at day 0 (before plating onto
dishes, 3 day of neurosphere culture), day 1 (the next day after plating
onto dishes), day 2, and day 6. (b) Protein levels of Rufy3, Tuj1, Sox2,
and NeuN during primary cortical NPC differentiation were determined
by western blot analysis. Arrows indicate the specific signal for NeuN.
(c) The expression of Rufy3 in mouse primary post-natal day 0 (P0)
cortical neurons were examined by double staining using antibodies
against Rufy3 (red) and Tuj1 (green). The upper panel shows images
of neurons with shorter outgrowth, while the bottom panel shows the
images of neurons with longer axons. (c’) is an enlarged image of the
inbox shown in the bottom panel of C for Rufy3 staining. (d) The sketch
marker) indicated that Rufy3 coexpressed with Tuj1 in the upper
layers of the cortex and in other brain regions where neurons
were abundant, but did not coexpress with Nestin (Fig. 1d). In
the mouse E18.5 cortex, cells with high Rufy3 expression were
distributed in the regions from the intermediate zone (IZ) to the
cortical plate (CP). The expression of Rufy3 exhibited identical
distribution to that of Tuj1 expression, but was different from
that of Nestin. Furthermore, results from double staining using
antibodies against Rufy3 and Map2 (a neuronal dendrite marker)
showed that Rufy3 proteins were also present in dendrites
containing Map2 proteins (Fig. 1e). These data clearly indicate
that Rufy3 is a brain-specific protein predominantly expressed in
the neuron-rich regions of the central nervous system.
Rufy3 is specifically expressed in mouse primary cortical
neurons and embryonic stem cell-derived neurons
To test whether Rufy3 is indeed a neuron-specific protein, we
examined the expression of Rufy3 in neurons differentiated
in vitro from cortical NPCs of E14.5 mouse embryos, as
neurogenesis becomes abundant in mouse embryos around
E13 (Gardette et al. 1982). When neurospheres, formed by
suspension culture were digested into single cells and plated
onto Matrigel-coated dishes, many NPCs appeared on day 1
of the plating (The last day of neurosphere suspension culture
was defined as differentiation day 0). From day 2 of the
plating, the NPCs began to differentiate into neurons
efficiently. Around day 6, neuronal death took place and
the cell number declined (Fig. 2a). Results of western blot
analysis showed that Rufy3 proteins were hardly detectable
at differentiation day 0, increasing from day 2 to day 4 and
decreasing at day 6. A low level of Tuj1 proteins was
observed at day 0, displaying a similar pattern as Rufy3 from
day 2 to day 6. Unlike Tuj1 and Rufy3, proteins of NeuN (a
mature neuron maker) were not detected until day 4 and
continued to increase at day 6, whereas Sox2 (a stem cell
marker) had a highest level at day 0 and decreased drastically
map of mouse embryonic stem cell (ESC) neural differentiation
process. After 7 days of embryoid body formation under a serum-free
condition (SFEB), embryoid bodies were plated onto Matrigel-coated
dishes and passaged every 3 days. (e) Expression patterns of different
markers during the mouse ESC neural differentiation process were
determined by qRT-PCR. The mRNA level in undifferentiated ESCs
was set as I. Three independent experiments were performed. Data
are shown as mean SD. (f) Expression levels of Rufy3 during the
mouse ESC neural differentiation process were examined by qRTPCR and western blot analyses. Data of mRNA levels are from three
independent experiments. One representative result from three independent western blot analyses is shown. (g) The cell types expressing
Rufy3 specifically were determined by immunofluorescence double
staining in mouse ESC-derived neural cells, using antibodies against
Rufy3 (red) and Nestin (green), or Tuj1 (green) or GFAP (green). The
Nucleus was stained by 4, 6-diamidino-2 phenylindole (DAPI). Scale
bars: 100 lm in (a); 50 lm in (c); 0.4 lm in C’; 25 lm in (g).
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
Rufy3 controls axon growth
(a)
(b)
(c)
(c’)
(f)
(d)
(g)
(e)
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after day 2 (Fig. 2b). This result suggests that Rufy3 might
be highly expressed in newly generated neurons with
decreased expression during neuronal maturation. Next, we
examined Rufy3 expression at a single-cell level using P0
mouse primary cortical neurons and found that Rufy3 was
widely expressed in all types of Tuj1+ cortical neurons. To
determine whether Rufy3 would be detected in the axon, in
addition to the cell body and dendrite, we carried out
immunostaining for the neurons with longer axons using
SMI 312 (a pan-axon marker) antibody to define axons. Our
result showed that Rufy3 could be detected in the axon
(Figure S3b). Taken together, Rufy3 proteins distributed in a
spotty fashion in the cell body as well as in the outgrowth of
primary mouse cortical neurons (Fig. 2c).
To obtain additional evidence that Rufy3 is a neuronspecific protein, we examined the expression pattern of
Rufy3 during neural differentiation of mouse ESCs using a
previously reported protocol (Watanabe et al. 2005), as
illustrated in Fig. 2d. The neural differentiation process was
characterized by the expression of various markers determined by real-time quantitative PCR (qRT-PCR). For
example, Nestin was highly expressed at differentiation days
10 and 13, and its expression decreased afterward. The
expression of Tuj1 reached to the peak at differentiation day
13, whereas the expression of glial markers Olig2 and Gfap
did not reach their maximum levels until the late stages of the
differentiation process (Fig. 2e). The sequential generation of
NPCs, neurons and glial cells from ESCs matched the
embryonic neural differentiation process. When expression
of Rufy3 was examined using the same neural differentiation
model, we found that it had an expression pattern identical to
that of Tuj1 (Fig. 2f, upper panel). The transcript level of
Rufy3 was barely detectable in undifferentiated ESCs,
reached its peak at differentiation day 13 and declined
afterward. Similarly, the protein level of Rufy3 was upregulated until day 16 of differentiation and then decreased
as the cells differentiated (Fig. 2f, lower panel). The identical
temporal expression pattern of Rufy3 and Tuj1 is consistent
with the notion that Rufy3 might be highly expressed in
neurons but not other cell types. To further confirm this
hypothesis, immunofluorescence staining was carried out in
mouse ESC-derived neural cells (Fig. 2g). We found that
Nestin and Rufy3-positive staining occurred exclusively in
the same cells when antibodies against Nestin and Rufy3
were used to doubly stain the cells. Moreover, Rufy3 was not
expressed in GFAP+ radial glia or astrocytes. Notably, every
Tuj1-positive neuron expressed Rufy3, indicating that Rufy3
was coexpressed with Tuj1 in neurons. Therefore, we clearly
demonstrate that Rufy3 is a neuron-specific protein.
Rufy3 controls the axonal growth and polarization process
in hippocampal neurons
The next question was the function of Rufy3 in neurons. We
addressed the question using both over-expression and RNA
interference (RNAi) strategies. First, constructs containing
the Rufy3-IRES-EGFP sequence or the IRES-EGFP control
sequence were transfected into mouse P0 hippocampal
neurons before plating. Two days later, the axon length of
EGFP+ neurons and the percentage of EGFP+ neurons with
multiple axons were measured. The morphology of neurons
was revealed by EGFP expression and the axon was defined
by SMI 312 staining (Fig. 3a). We found that the axonal
length of neurons over-expressing Rufy3 was significantly
longer than that of neurons transfected with a control plasmid
(n = three independent cultures, 128 neurons examined;
p < 0.05) (Fig. 3b). Moreover, the percentage of neurons
with multiple axons seemed reduced by the expression of
exogenous Rufy3, although the difference was not statistically significant (Fig. 3c). Thus, we concluded that overexpression of Rufy3 enhanced axonal growth. Second, we
silenced Rufy3 expression by transfection of hippocampal
neurons with two shRNA plasmids, which targeted two
different regions of the Rufy3 mRNA (Ri-1#, Ri-2#),
respectively, and contained a separate cassette for constitutive EGFP expression. The silencing efficiency of the shRNA
plasmids was verified by western blot analysis of endogenous Rufy3 protein levels in neurons (Figure S3a). In
hippocampal neurons transfected with both shRNA plasmids,
knockdown of Rufy3 expression led to significant shortening
of axons (n = three independent cultures; 96 neurons
transfected with Ri-1# plasmid, p < 0.01, when compared
with the control plasmid; 91 neurons transfected with Ri-2#
plasmid, p < 0.01, when compared with the control plasmid)
(Fig. 3d and e). Moreover, the percentage of EGFP+ neurons
bearing more than one (two or three) axons increased in
Rufy3-knocked down neurons (p < 0.001) (Figs. 3d and f).
Therefore, appropriate expression of Rufy3 is required for
both establishment of normal polarity and maintenance of
axonal growth in mouse hippocampal neurons.
Rufy3 interacts and colocalizes with F-actin-binding
proteins Fascin and Drebrin in neuronal growth cones
Having defined the role of Rufy3 in the axonal growth of
mouse hippocampal neurons, we went on to investigate how
Rufy3 influenced axon growth at a molecular level. As Rufy3
contains RUN and coil–coil domains, which could mediate
protein–protein interactions, we assumed that identification
of Rufy3-associated proteins in neurons would facilitate our
understanding how it executes its functions. To this end,
immunoprecipitation experiments were carried out using
tissue extract of the whole-mouse P0 brain and the Rufy3
antibody to purify endogenous Rufy3-associated protein
complexes. Precipitated proteins were separated on SDS–
PAGE gels, which were then cut off for mass spectromic
(MS) analysis (Supplementary Information, Figure S4). The
name of proteins specifically precipitated by Rufy3 antibodies is listed in Table 1. Interestingly, cytoskeletal proteins
such as Spectrin, Filamin A, Ankyrin-2, Fascin, and Drebrin
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
Rufy3 controls axon growth
(a)
685
(d)
(b)
(c)
(e)
(f)
Fig. 3 Rufy3 plays an important role in axon morphogenesis in mouse
hippocampal neurons. (a) The morphology of mouse hippocampal
neurons transfected with control (Ctrl) and Rufy3 over-expression
(Rufy3 OE) plasmids. All plasmids contained a CAG promoter-driven
EGFP cassette. Cells were stained with SMI312 antibody to label
axons (red). (b) The axon length of mouse hippocampal neurons
transfected with the Rufy3 OE plasmid were compared with that of
control cells. (c) The percentage of mouse hippocampal neurons with
multiaxons was compared between Rufy3 OE and control cells. (d)
The morphology of mouse hippocampal neurons transfected with
control and Rufy3 shRNA plasmids (Ri-1# and Ri-2#). The plasmids
contained a PGK promoter-driven EGFP expression cassette. (e) The
axon length of mouse hippocampal neurons transfected with Rufy3
shRNA plasmids was compared with that of control cells (Ri-1# with
control, Ri2# with control, respectively). (f) The percentage of mouse
hippocampal neurons with multiaxons was compared between control
and Rufy3 shRNA cells (Ri-1# with control, Ri2# with control,
respectively). *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars:
75 lm. Data are shown as mean SD.
were enriched in the list. Consistent with that result, the gene
ontology (GO) analysis also showed that Rufy3-associated
proteins might be involved with the control of cytoskeletons
(Fig. 4a). The finding suggests that Rufy3’s role in neuronal
morphogenesis may be related to the regulation of cytoskeletal organization. Of these proteins, we focused on Fascin
and Drebrin, as both are known F-actin-binding proteins and
are involved in the growth of neuronal cones.
Specific interactions between Rufy3 and Fascin or Drebin
were verified by Co-IP assays using tissue extracts from the
P0 mouse brain. As shown in Fig. 4b, Drebrin antibodies
could pull down Rufy3 and Fascin proteins in addition to
Drebrin proteins; proteins of Fascin, Rufy3, and Drebrin
could be detected in the complexes precipitated by Rufy3
antibodies; and Fascin antibodies coprecipitated Drebrin and
Rufy3 as well as Fascin proteins. As negative controls, IgG
could not bring down any of these three proteins. Therefore,
we show here for the first time that endogenously expressed
Rufy3 interacts with Fascin and Drebrin specifically in the
brain cells under physiological conditions.
To further characterize the interaction between Rufy3 and
Fascin or Drebrin, we over-expressed Rufy3 and HA-tagged
Fascin or HA-tagged Drebrin in 293T cells. Interestingly, we
were unable to detect the interaction between exogenously
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
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Table 1 The list of Rufy3 interacting proteins identified by mass spectrometry
Accession
Name
Peptides
(95%)
sp|P16546|SPTA2_MOUSE
sp|P57780|ACTN4_MOUSE
sp|Q7TPR4|ACTN1_MOUSE
sp|Q62261|SPTB2_MOUSE
sp|Q8BTM8|FLNA_MOUSE
sp|P46660|AINX_MOUSE
sp|P63260|ACTG_MOUSE
sp|P60710|ACTB_MOUSE
sp|Q61553|FSCN1_MOUSE
sp|Q9D394|RUFY3_MOUSE
sp|Q9WUM4|
COR1C_MOUSE
sp|Q8BH44|COR2B_MOUSE
sp|Q9QXS6|DREB_MOUSE
sp|Q99K48|NONO_MOUSE
Spectrin alpha chain, brain OS = Mus musculus GN = Sptan1 PE = 1 SV = 4
Alpha-actinin-4 OS = Mus musculus GN = Actn4 PE = 1 SV = 1
Alpha-actinin-1 OS = Mus musculus GN = Actn1 PE = 1 SV = 1
Spectrin beta chain, brain 1 OS = Mus musculus GN = Sptbn1 PE = 1 SV = 2
Filamin-A OS = Mus musculus GN = Flna PE = 1 SV = 5
Alpha-internexin OS = Mus musculus GN = Ina PE = 1 SV = 2
Actin, cytoplasmic 2 OS = Mus musculus GN = Actg1 PE = 1 SV = 1
Actin, cytoplasmic 1 OS = Mus musculus GN = Actb PE = 1 SV = 1
Fascin OS = Mus musculus GN = Fscn1 PE = 1 SV = 4
Protein RUFY3 OS = Mus musculus GN = Rufy3 PE = 1 SV = 1
Coronin-1C OS = Mus musculus GN = Coro1c PE = 1 SV = 2
27
26
16
16
10
9
8
8
6
6
3
sp|Q8C8R3|ANK2_MOUSE
sp|Q62167|DDX3X_MOUSE
sp|Q62095|DDX3Y_MOUSE
sp|Q9QXZ0|MACF1_MOUSE
sp|P16381|DDX3L_MOUSE
sp|Q8CAQ8|IMMT_MOUSE
sp|Q99KE1|MAOM_MOUSE
sp|Q61656|DDX5_MOUSE
sp|Q7TMM9|TBB2A_MOUSE
sp|Q9CWF2|TBB2B_MOUSE
sp|P68372|TBB4B_MOUSE
Coronin-2B OS = Mus musculus GN = Coro2b PE = 2 SV = 2
Drebrin OS = Mus musculus GN = Dbn1 PE = 1 SV = 4
Non-POU domain-containing octamer-binding protein OS = Mus musculus GN = Nono PE = 1
SV = 3
Ankyrin-2 OS = Mus musculus GN = Ank2 PE = 1 SV = 2
ATP-dependent RNA helicase DDX3X OS = Mus musculus GN = Ddx3x PE = 1 SV = 3
ATP-dependent RNA helicase DDX3Y OS = Mus musculus GN = Ddx3y PE = 1 SV = 2
Microtubule-actin cross-linking factor 1 OS = Mus musculus GN = Macf1 PE = 1 SV = 2
Putative ATP-dependent RNA helicase Pl10 OS = Mus musculus GN = D1Pas1 PE = 1 SV = 1
Mitochondrial inner membrane protein OS = Mus musculus GN = Immt PE = 1 SV = 1
NAD-dependent malic enzyme, mitochondrial OS = Mus musculus GN = Me2 PE = 2 SV = 1
Probable ATP-dependent RNA helicase DDX5 OS = Mus musculus GN = Ddx5 PE = 1 SV = 2
Tubulin beta-2A chain OS = Mus musculus GN = Tubb2a PE = 1 SV = 1
Tubulin beta-2B chain OS = Mus musculus GN = Tubb2b PE = 1 SV = 1
Tubulin beta-4B chain OS = Mus musculus GN = Tubb4b PE = 1 SV = 1
3
3
3
2
2
2
2
2
1
1
1
1
1
1
The proteins in the Rufy3 (Rufy3 antibody IP) group having a value at a 95% confidence interval at least two folds greater than that in the control
(IgG IP) group were selected and are listed.
expressed Rufy3 and HA-tagged Drebrin in 293T cells,
although the specific interaction between Rufy3 and HAFascin was readily observed (Fig. 4c). This phenomenon
suggests that the specific interaction between endogenous
Rufy3 and Drebrin detected in neurons might not be direct, but
mediated through factor (s) absent from 293T cells; or the
interaction only occurs under certain conditions. As exogenously expressed Rufy3 and Fascin could interact, we next
determined the domain of Rufy3 mediating its interaction with
Fascin. Flag-tagged full-length Rufy3, the RUN domain in its
N-terminus, or the coil–coil domain in its C-terminus was
coexpressed with or without HA-tagged Fascin in 293T cells
and Co-IP experiments were performed (Fig 4d). Our results
indicated that Fascin interacted with the full-length and the Nterminus of Rufy3, but not with the C-terminus (Fig. 4e). As
the RUN domain is the only known domain in the N-terminus
of Rufy3, we proposed that it was the RUN domain of Rufy3
responsible for its association with Fascin.
Both Drebrin and Fascin have been reported to be
distributed in growth cones (Edwards and Bryan 1995;
Geraldo et al. 2008). Moreover, our study indicated that
they were also highly expressed during neural differentiation of ESCs and in the developing brain (Figure S2c and
d). However, whether they are codistributed with Rufy3 in
growth cones is unknown. To answer this question,
immunofluorescence staining was performed in mouse P0
hippocampal neurons using the specific Rufy3 antibody.
We found that Drebrin was mainly expressed at the
transitional domain, as reported previously (Geraldo and
Gordon-Weeks 2009), whereas Rufy3 and Fascin were
distributed primarily at filopodia and lamellipodia,
although they were also seen at the transitional domain
and central domain of the growth cone (Fig. 4f). Rufy3
staining appeared punctate and coincided with Fascin
entirely in the whole growth cone, only partially overlapping with Drebrin at the transitional domain. In addition,
Rufy3 displayed a similar distribution pattern to F-actin at
the peripheral and transitional domains (Fig. 4g), while
Drebrin was partially coexpressed with F-actin at the
transitional domain. Our results suggest that Rufy3 may be
a new member of the F-actin binding and bundling protein
family.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
Rufy3 controls axon growth
(a)
(c)
687
(b)
(f)
(d)
(g)
(e)
Fig. 4 Rufy3 interacts with Drebrin and Fascin, colocalizing with F-actin
in the growth cone of mouse hippocampal neurons. (a) The GO analysis
for Rufy3 interacting proteins identified by mass spectrometry based
on the biological process. The p-value was converted into –log10. The
biological process with a smaller p-value is more significant than others.
(b) Endogenous coimmunoprecipitation (Co-IP) between Rufy3 and
Drebrin or Fascin using extracts of post-natal day 0 (P0) mouse brains.
The symbol * indicates the heavy chain of IgG. (c) Exogenous Co-IP
between Rufy3 and Fascin in 293T cells. (d) A sketch map of different
domains for Rufy3 proteins. These domains were constructed into a
Flag-pPy plasmid and expressed in 293T cells. (e) Protein interactions
between different domains of Rufy3 and Fascin in 293T cells. The cDNA
of full-length Fascin was constructed into a HA tagged plasmid. The
symbol # indicates non-specific signals in the western blot analysis. The
arrows indicate specific HA-Fascin signals. The symbol * indicates the
heavy chain of IgG. (f) Subcellular distribution of Drebrin, Rufy3, and
Fascin in growth cones of mouse hippocampal neurons was detected by
immunofluorescence staining. The neurons were double stained with
Rufy3 (green) and Drebrin (red) or Rufy3 (red) and Fascin (green)
antibodies. Growth cone and the distal part of axon are shown. (g) The
localization of Drebrin, Rufy3, and Fascin was compared to that of
F-actin in growth cones. The hippocampal neurons were double stained
with antibodies against Drebrin (green) and F-actin (red), or Fascin
(green) and F-actin (red) or Rufy3 (green) and F-actin (red). Only growth
cones are shown in images. Scale bars: 10 lm. IB: immunoblotting. IP:
immunoprecipitation.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
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Z. Wei et al.
Rufy3 and F-actin are mutually dependent for normal
distribution in the axonal growth cone
As Rufy3 colocalized with F-actin in the growth cone, we
tested whether disruption of F-actin would affect distribution
of Rufy3 in growth cones by treating cultured hippocampal
neurons at day 2 for 2 h with 1 lM of cytochalasin D
(CytoD), an inhibitor of actin polymerization (Nair et al.
2008). After CytoD treatment, cultured neurons formed
axons without large growth cones, as reported previously
(Bradke and Dotti 1999). Simultaneously, F-actin contents
decreased drastically in the peripheral and transitional
domains, with a small amount of F-actin scattered along
the edges of filopodia and lamellipodia. Interestingly, CytoD
treatment also led to a decrease in Rufy3 staining, which
exhibited a distribution pattern identical to F-actin (Fig. 5a),
indicating that the localization of Rufy3 in growth cones
depended on the F-actin organization. In addition, disruption
of F-action brought about an almost complete loss of Fascin
and Drebrin in the transitional and central domains, with a
small amount of these two proteins detected at the edge of the
growth cone (Fig. 5b and c), in accordance with previous
reports (Cohan et al. 2001; Mizui et al. 2009). Notably, Factin staining at the edge of growth cones invariably
colocalized with the staining of Rufy3, Drebrin, and Fascin
after CytoD treatment. These observations indicate that
Rufy3 is closely associated with F-actin in growth cones, as
are Drebrin and Fascin.
We then asked whether appropriate expression of Rufy3
was required for normal organization of F-actin. Hippocampal neurons of P0 mice were nucleofected with Rufy3
(a)
(b)
(c)
Fig. 5 Normal distribution of Rufy3 in the
growth cone relies on the polymerization of
F-actin. Mouse hippocampal neurons used
in (a–c) were cultured for 2 days and then
treated with 1 lM of cytochalasin D (CytoD)
for 2 h before fixation. Typical bright field
and immuno- fluorescence staining images
are shown. Images of double staining of
Rufy3 (green) and F-actin (red) with or
without CytoD treatment are shown in (a).
Arrows indicate some signals of F-actin and
Rufy3 after adding CytoD. Images of double
staining of Fascin (green) and F-actin (red)
are shown in (b). Arrows indicate some
signals of F-actin and Drebrin after adding
CytoD. Images of double staining of Drebrin
(green) and F-actin (red) are shown in (c).
Arrows indicate some signals of F-actin and
Fascin after adding CytoD. Scale bars:
10 lm.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
Rufy3 controls axon growth
over-expression plasmid (OE) or shRNA plasmids (Ri-1#,
Ri-2#), both containing a constitutive EGFP expression unit.
The EGFP expression cassette was used to illustrate the
morphology of the growth cones. Over-expression of Rufy3
did not alter the localization of F-actin (Fig. 6a). However,
knockdown of Rufy3 reduced F-actin staining intensity in the
transitional domain and F-actin appeared to concentrate at the
edge of the growth cone (Fig. 6b). Thus, Rufy3 played an
important role in the normal function of F-actin and the
proper structure of growth cones.
Rufy3 is required for the normal localization of Drebrin and
Fascin in axonal growth cones
Given that normal expression of Rufy3 is crucial for proper
organization of F-actin in axonal growth cones and that
disruption of F-action structure could affect the distribution
of Fascin and Drebrin, we anticipated that aberrant expression of Rufy3 would also alter the localization of Fascin and
Drebrin. To verify this assumption, we conducted the same
over-expression and RNAi experiments to test whether
Rufy3 could affect the distribution of Fascin and Drebrin.
Over-expression of Rufy3 seemed to increase the staining
689
intensity of Drebrin in the growth cone, which extended to
the peripheral domain from its normal position in the central
and transitional domains (Fig. 7a). In contrast, the localization of Fascin was not significantly affected by Rufy3 overexpression (Fig. 7b). However, when the expression of
Rufy3 was knocked down, the intensity of Fascin staining
decreased with restricted localization at the edge of the
growth cone (Fig. 7d), while the distribution pattern and
staining intensity of Drebrin did not change (Fig. 7c). These
data clearly demonstrate that there are functional interactions
among Rufy3, Fascin, Drebrin, and F-actin, and that Rufy3 is
an important player in cytoskeletal organization during axon
development in mouse neurons.
Discussion
In this study, we proposed that Rufy3 is a new member of Factin-associated proteins specifically expressed in mouse
neurons and important for neural axonal morphogenesis.
Rufy3 colocalized with F-actin entirely in the peripheral and
transitional domains of axonal growth cones in mouse
hippocampal neurons. Importantly, the distribution of Rufy3
(a)
(b)
Fig. 6 Normal distribution of F-actin in the
growth cone of mouse hippocampal neurons
requires the presence of Rufy3. (a) Typical
bright field images and images of double
staining of F-actin (red) and enhanced green
fluorescent protein (EGFP) (green) in mouse
hippocampal neurons transfected with
control (ctrl) or Rufy3 over-expression
plasmid (Rufy3 OE) are shown. (b) Typical
bright field images and images of double
staining of F-actin (red) and EGFP (green) in
mouse hippocampal neurons transfected
with control (ctrl) or Rufy3 shRNA plasmid
(Ri-1#, Ri-2#). Arrows indicate some signals
of F-actin after Rufy3 shRNA plasmids were
transfected. Scale bars: 9 lm.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
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Z. Wei et al.
Fig. 7 Rufy3 controls distribution of Fascin and Drebrin in the growth
cone of mouse hippocampal neurons. (a) Typical bright field images
and images of double staining of Drebrin (red) and enhanced green
fluorescent protein (EGFP) (green) in mouse hippocampal neurons
transfected with control (ctrl) or Rufy3 over-expression plasmid (Rufy3
OE). Arrows indicate some signals of Drebrin after Rufy3 overexpression plasmids were transfected. (b) Typical bright field images
and images of double staining of Fascin (red) and EGFP (green) in
mouse hippocampal neurons transfected with control (ctrl) or Rufy3
over-expression plasmid (Rufy3 OE). (c) Typical bright field images
and images of double staining of Drebrin (red) and EGFP (green) in
mouse hippocampal neurons transfected with control (ctrl) or Rufy3
shRNA plasmid (Ri-1#, Ri-2#). (d) Typical bright field images and
images of double staining of Fascin (red) and EGFP (green) in mouse
hippocampal neurons transfected with control (ctrl) or Rufy3 shRNA
plasmid (Ri-1#, Ri-2#). Arrows indicate some signals of Fascin after
Rufy3 shRNA plasmids were transfected. Scale bars: 10 lm.
(a)
(b)
(c)
(d)
and F-actin in growth cones was dependent on each other.
Actin bundles are important not only for the formation of
growth cones through supporting filopodial extension but also
for cone motility by controlling protrusion of lamellipodia and
filopodia at the leading edge. The bundling of F-actin is
regulated by different actin binding and bundling proteins
(Bartles 2000). Thus, identification of Rufy3 as a new
F-actin-associated protein is significant for understanding
how axons form and elongate. In particular, we found that
Rufy3 was exclusively expressed in neurons of mouse brain
tissues, but not in NPCs or glia cells, suggesting its unique role
in neuronal development. Indeed, in this study, Rufy3 overexpression enhanced the axon length significantly and reduced
the percentage of neurons with multiple axons. Conversely,
Rufy3 knockdown reduced the axonal length substantially and
increased the neurons with surplus axons in mouse primary
hippocampal neurons, suggesting that Rufy3 is a crucial player
for controlling axon formation and elongation. Consistent with
our finding, a previous study showed that Singar 1 was
important for suppressing surplus axons in rat hippocampal
neurons (Mori et al. 2007). However, in that study, silencing
Singar 1 did not alter the axonal length, and Singar 1 overexpression did not affect the axon formation or axon length
during normal polarization process. Therefore, it appears that
Rufy3 proteins from the rat and mouse may play similar and
different roles in neuronal morphogenesis. Our discovery of
Rufy3 as a neuron-specific protein functionally relevant to Factin places Rufy3 at an important position in the control of
axon formation and elongation.
Additional evidence to support a close relationship
between Rufy3 and F-actin came from the identification of
Rufy3-associated protein complexes. The highly specific
Rufy3 antibody developed in our laboratory provided us an
opportunity to purify endogenous proteins interacting with
Rufy3 in a physiological context. There were several
cytoskeleton-associated proteins in the Rufy3 protein complexes. Among these proteins, we verified the specific
interactions between Rufy3 and Drebrin or Fascin. Similar
to Rufy3, Drebrin has been shown to be expressed throughout neurons. However, its highest levels are in the cell soma
and growth cone, where it localizes to the transitional domain
and the proximal region of filopodia where microtubules
insert (Geraldo et al. 2008). Geraldo et al. demonstrated that
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 678--692
Rufy3 controls axon growth
the specific interaction between the Drebrin bound to the
proximal F-actin bundle of filopodia and microtubule and
plus-tip protein EB3 located at the tip of microtubules
invading filopodia is essential for growth cone formation and
neurite extension. We found that Rufy3 formed protein
complexes with Drebrin in neurons, but not in non-neural
293T cells, suggesting that the interaction between these two
proteins might be indirect or occur only under certain
conditions. Interestingly, over-expression of Rufy3 caused
Drebrin proteins to localize to a more peripheral region,
while Rufy3 knockdown did not affect its distribution. As
over-expression of Rufy3 did not alter localization of F-actin
and Fascin, it is unlikely that the aberrant localization of
Drebrin mediated by Rufy3 over-expression was through Factin or Fascin. In fact, we found that the distribution of
Rufy3 in growth cones resembled Fascin. Fascin was shown
to incorporate into F-actin from the beginning of growth cone
formation and to localize to and associate with radially
oriented actin bundles in lamellipodia and filopodia except at
the proximal part adjacent to the central domain, where actin
disassembling takes place. The changes in Fascin’s actinbundling activity, which is inhibited by phosphorylation,
could alter the actin bundle organization (Cohan et al. 2001).
Similar to Fascin, Rufy3 was found in the cell body, axon,
and growth cones. Punctate Rufy3 staining could be
observed in both lamellipodia and filopodia, entirely colocalizing with Fascin and partially with Drebrin. Co-IP results
further supported the close relationship between Rufy3 and
Fascin. Notably, knockdown of Rufy3 reduced Fascin and Factin expression in the proximal part of growth cones,
although Drebrin expression was not affected by the
silencing of Rufy3. Thus, we favor the notion that Rufy3
plays an important role in the maintenance of F-actin
structure and axonal growth, probably through its interaction
with Fascin. Nevertheless, we do not rule out the possibility
that other proteins identified in the Rufy3 protein complexes
are also involved in the regulatory effect of Rufy3 on axon
elongation. Moreover, it remains unclear whether Rufy3
suppresses the surplus axons and regulates the axon growth
through the same or different mechanisms. It is obvious that
the normal distribution of Drebrin, Fascin, and Rufy3 all
require the presence of F-actin. Further investigation is
needed to understand how the three proteins interact, in
particular how Rufy3 influences Fascin.
During human embryo development, the expression of
RUFY3 was found to increase during embryonic days 20–32,
with an expression pattern similar to those of neural markers
such as TUJ1 and MAP2, but different from those of
mesoendoderm markers, such as GATA4 and GATA6. This
suggests that RUFY3 may also be expressed in neural tissues
of human embryos. Considering the function of Rufy3 in the
mouse and the rat, the function of Rufy3 in human
embryonic development should be studied using human
ESC neural differentiation as an in vitro model. In addition,
691
actin-binding proteins are known to regulate the morphological changes of neurons in neurodegenerative diseases. For
instance, Drebrin has been implicated in Alzheimer’s
diseases (Shim and Lubec 2002). It will be interesting to
examine whether Rufy3 is also associated with the development of human neural disorders.
Acknowledgments and conflict of interest
disclosure
We would like to thank Dr Zhenge Luo for his helpful discussion
and Dr Yongchun Yu for his technical support in the brain section
experiments. In addition, authors thank Erbo Xu for his editing of
the manuscript. This study was supported by grants from the
Chinese Academy of Sciences (XDA01010102), the National
Natural Science Foundation (91019023), and the National High
Technology Research and Development Program of China
(2010CB945200, 2011DFB300100, 2011CB965101, and 2009CB
941103). No competing interests exist.
All experiments were conducted in compliance with the ARRIVE
guidelines.
Supporting information
Additional supporting information may be found in the online
version of this article at the publisher's web-site:
Figure S1. The expression levels of RUFY3 and other markers
during 2 early human embryonic development.
Figure S2. Expression of Rufy3 family members and the
expression pattern of Drebrin and Fascin in different tissues and
different developmental stages of mouse brains.
Figure S3. The efficiency of Rufy3 shRNA plasmids and
immunostaining of Rufy3 and SMI 312.
Figure S4. The SDS–PAGE gel for mass spectrum analysis.
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