Requirements for Hirano Body Formation

Paul Grifn,a* Ruth Furukawa,a Cleveland Piggott,a* Andrew Maselli,b Marcus Fechheimera
Department of Cellular Biology, University of Georgia, Athens, Georgia, USAa; Department of Biological Sciences, Chicago State University, Chicago, Illinois, USAb

Hirano bodies are paracrystalline F-actin-rich structures associated with diverse conditions, including neurodegeneration and
aging. Generation of model Hirano bodies using altered forms of Dictyostelium 34-kDa actin-bundling protein allows studies of
their physiological function and mechanism of formation. We describe a novel 34-kDa protein mutant, E60K, with a point muta-
tion within the inhibitory domain of the 34-kDa protein. Expression of E60K in Dictyostelium induces the formation of model
Hirano bodies. The E60K protein has activated actin binding and is calcium regulated, unlike other forms of the 34-kDa protein
that induce Hirano bodies and that have activated actin binding but lack calcium regulation. Actin filaments in the presence of
E60K in vitro show enhanced resistance to disassembly induced by latrunculin B. Actin filaments in model Hirano bodies are
also protected from latrunculin-induced depolymerization. We used nocodazole and blebbistatin to probe the role of the micro-
tubules and myosin II, respectively, in the formation of model Hirano bodies. In the presence of these inhibitors, model Hirano
bodies can form but are smaller than controls at early times of formation. The ultrastructure of model Hirano bodies did not
reveal any major difference in structure and organization in the presence of inhibitors. In summary, these results support the
conclusion that formation of model Hirano bodies is promoted by gain-of-function actin filament bundling, which enhances
actin filament stabilization. Microtubules and myosin II contribute to but are not required for formation of model Hirano
bodies.

A ctin is a major cytoskeletal protein found in all eukaryotic

cells. Actin can be found in two forms: monomeric (G-actin)
and filamentous (F-actin). The populations of both G-actin and
F-actin are controlled and regulated by a large number of actin-
interacting proteins that modulate the assembly and disassembly
of actin filaments (1). For example, unpolymerized actin pools are
maintained primarily through G-actin-binding proteins that se-
quester monomeric actin (2). In contrast, F-actin-binding pro-
teins help to stabilize F-actin structures and to cross-link and/or
bundle actin filaments into elaborate multimolecular structures
(3). The actin cytoskeleton is involved in many important cellular
processes, such as cell motility, cell shape, cell signaling, and in-
tracellular trafficking. Tight regulation of the actin cytoskeleton is
important to proper cellular function.
Cytoplasmic aggregations containing actin and actin-binding
proteins are associated with a number of neurodegenerative dis-
eases and can assemble as either actin/cofilin rods or Hirano bod-
ies (4, 5). Hirano bodies are large, eosinophilic, actin-rich intra-
cytoplasmic structures that contain paracrystalline arrays of
F-actin (6) and actin-binding proteins (7). Hirano bodies have
been described in postmortem tissues and are found primarily in
the CA1 area of Ammons horn of the hippocampus (810). Hi-
rano bodies have been found to be associated with a number of
different neurodegenerative diseases, such as Alzheimers disease
(1113), Picks disease (9, 14), and Guam amylotrophic lateral
sclerosis and parkinsonism-dementia complex (8). Hirano bodies
have also been associated with chronic alcoholism (15), as well as
general aging (5). Despite a large body of literature describing the
structure, incidence, and antigens associated with Hirano bodies,
the mechanism by which Hirano bodies are formed and their re-
lationship to disease remain largely unexplored.
A cellular system enables the formation of model Hirano bod-
ies for studies of the formation and physiological consequences of
these structures. Model Hirano bodies were induced to form in
the cellular slime mold Dictyostelium discoideum and mammalian
cells by expression of altered forms of a 34-kDa actin cross-linking
protein (1618). The 34-kDa actin-binding protein is one of many
actin cross-linking proteins found in Dictyostelium (3, 19) and has
been biochemically characterized as a calcium-sensitive actin-
bundling protein (2022). Molecular dissection of recombinant
forms of the 34-kDa protein has revealed three actin-binding sites
(residues 1 to 123, 193 to 254, and 279 to 295), as well as an
N-terminal inhibitory domain (residues 1 to 76) that is proposed
to regulate 34-kDa protein binding to F-actin (23, 24). A purified
C-terminal fragment (CT) (residues 124 to 295) was characterized
in vitro and found to possess a calcium-insensitive enhanced F-ac-
tin affinity (23, 24). Therefore, it has been proposed that CT-
induced model Hirano body formation may be the result of in-
creased F-actin binding and loss of calcium regulation (16).
Further studies using additional mutated forms of the 34-kDa
protein are consistent with the conclusion that activation of actin
binding and loss of calcium regulation are biochemical properties
invariably associated with induction of model Hirano bodies (17,
25). On the cellular level, this model system was used to demon-
strate that Hirano body formation proceeds by formation of small
aggregates, followed by consolidation into a single large Hirano
body (26). Degradation of Hirano bodies in this model is complex
and involves the proteasome, autophagy, and secretion to the ex-
tracellular milieu (27).
Received 17 February 2014 Accepted 6 March 2014
Published ahead of print 14 March 2014
Address correspondence to Ruth Furukawa, furukawa@uga.edu.
* Present address: Cleveland Piggott, University of North Carolina, Chapel Hill,
North Carolina, USA; Paul Griffin, Meridian Life Sciences, Memphis, Tennessee,
USA.
Copyright 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/EC.00044-14

May 2014 Volume 13 Number 5 Eukaryotic Cell p. 625 634 ec.asm.org 625
Grifn et al.
The goal of this study was to investigate the formation of
Hirano bodies by probing both the biochemical or structural fea-
tures required for the formation of Hirano bodies and the roles of
other cellular components. We describe a novel mutant form of
the 34-kDa protein (E60K) that has a point mutation at amino
acid position E60 in the proposed inhibitory domain. Studies of
E60K show that although it retains calcium-sensitive F-actin bind-
ing, it has a higher F-actin-binding affinity than the wild-type
34-kDa protein and induces formation of highly ordered
paracrystalline model Hirano bodies in Dictyostelium. In addition,
using the actin-depolymerizing drug latrunculin B (latB), we
demonstrate that depolymerization of actin filaments in vitro and
model Hirano bodies in living cells is severely inhibited by the
presence of the E60K protein. At the cellular level, neither depo-
lymerization of microtubules with nocodazole nor inhibition of
myosin II with blebbistatin induces a major change in the ultra-
structure of Hirano bodies. However, there was a discernible dif-
ference in the rate of formation of aggregates that grow into large
model Hirano bodies in the presence of either nocodazole or bleb-
bistatin. These observations support the hypothesis that both en-
hanced filament cross-linking and the inhibition of normal actin
filament turnover lead to an accumulation of F-actin that is fur-
ther organized into the highly ordered arrays that comprise Hi-
rano bodies.
MATERIALS AND METHODS
Plasmids. The E60K mutant form of the 34-kDa actin-bundling protein
in pVEII was generated serendipitously during construction of pVEII-34
kDa-EGFP (27). For recombinant-protein expression in bacteria, the
E60K cDNA was amplified from the pVEII-E60K-EGFP plasmid using
PCR and cloned into the BamHI site of pET-15b (EMD Millipore). The
plasmids were sequenced to verify fidelity.
Actin. G-actin was prepared from rabbit skeletal muscle acetone pow-
der, purified by Sephadex G-150 chromatography (28, 29), and main-
tained in G-actin buffer (2 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM
ATP, 0.2 mM dithiothreitol [DTT], and 0.02% NaN3) at 4C for up to 1
week with daily buffer changes. After 1 week, the G-actin was subjected to
a cycle of polymerization in high-salt buffer (50 mM KCl, 1 mM ATP, and
1 mM MgCl2) and depolymerization by dialysis against G-actin buffer
before maintenance in G-actin buffer.
Bacterial expression and purification of recombinant proteins. Ex-
pression and purification of the wild-type 34-kDa protein and the mutant
E60K protein in pET15b were performed as previously described for the
recombinant wild-type 34-kDa protein (30). The proteins were purified
to homogeneity as assessed by SDS-PAGE, followed by Coomassie blue
stain. The protein concentrations were determined by the bicinchoninic
acid (BCA) method (31), using bovine serum albumin as the protein
standard. All purified recombinant proteins were dialyzed against 2 mM
PIPES [piperazine-N,N=-bis(2-ethanesulfonic acid)], pH 7.0, 50 mM KCl,
0.2 mM DTT, 0.03% NaN3 and stored at 80C.
F-actin cosedimentation assays. High-speed F-actin cosedimenta-
tion assays were performed as previously described (20, 21). The F-actin
polymerization conditions were 20 mM PIPES, pH 7.0, 50 mM KCl, 1 mM
MgCl2, 1 mM ATP, 5 mM EGTA, 0.2 mM DTT. Evaluation of calcium
sensitivity was performed by the absence or presence of 4.5 mM CaCl2 in
the previous buffer. The free calcium ion concentrations were calculated
using maxchelator.stanford.edu with NIST database 46, vol.8, at pH 7 and
25C. The high-calcium and low-calcium conditions correspond to pH 7. The cells were permeabilized with acetone (20C) for 2 min. The
free calcium ion concentrations of 3.1 M and 1.4 108 M, respectively.
The solutions were centrifuged in a Beckman airfuge at 23 lb/in2
(115,000 g) for 30 min. The supernatant and pellet samples were col-
lected and analyzed by SDS-PAGE and Coomassie blue staining. Protein
626 ec.asm.org
bands were quantified using scanning densitometry (Molecular Dynam-
ics Laser Scanning Densitometer).
Latrunculin B (Sigma-Aldrich) cosedimentation assays were per-
formed by polymerizing 3 M G-actin in the presence or absence of either
3 M E60K or wild-type 34-kDa protein for 2 h at room temperature.
After polymerization, latB was added to a final concentration of 4.5 M
and incubated for either 2 h or 24 h prior to the cosedimentation assay
described above. Cosedimentation assays were performed a minimum of
three times.
Dictyostelium culture growth. Axenic strains of Dictyostelium
(E60K/AX2, E60K/abpB, E60K/atg5, and atg5) were routinely main-
tained in axenic cultures with shaking at 150 rpm in HL-5 medium (32) at
20C. Inducible expression of either the wild-type 34-kDa protein or the
E60K mutant protein fused to green fluorescent protein (GFP) was
achieved by utilizing the discoidin promoter in pVEII (33) so that protein
expression could be suppressed or induced by the addition or removal,
respectively, of 1 mM folate from the growth medium in cells lacking the
34-kDa protein (abpB). Mutant Dictyostelium bearing a disruption of
the autophagy gene atg5 (atg5) were a generous gift from Rich Kessin,
Columbia University, and have been described previously (34).
To induce expression of E60K and formation of model Hirano bodies,
cell cultures were grown to 1.0 106 cells/ml in the presence of folate and
harvested by centrifugation (800 g; 5 min at room temperature). The
cells were washed 2 times with Sorensens phosphate buffer, pH 6.1, to
remove the folate to induce E60K expression and resuspended at 5 105
cells/ml in HL5 medium with the appropriate antibiotics for the strain.
Ten milliliters of cell suspension was used to inoculate 100-mm petri
dishes. The cells were incubated at 20C for 16 to 20 h after plating on
coverslips. Under these growth conditions, 80% of pVEII-E60K-EGFP
cells contained at least 1 Hirano body (data not shown).
For expression experiments performed in the presence or absence of
either nocodazole or blebbistatin, cells were harvested approximately 24 h
prior to the start of induction; washed twice with Sorensens phosphate
buffer, pH 6.1, and twice with HL-5; and plated at 3.5 105 cells in HL-5
on microscope coverslips or in 6-well tissue culture-treated plates to in-
duce the expression of E60K-GFP. Nocodazole (Sigma-Aldrich) and bleb-
bistatin (Sigma-Aldrich) were present at final concentrations of 10 g/ml
and 100 M, respectively, and cells were examined at 3.5, 5.5, 7.5, and 24
h after induction of protein expression and in the presence or absence of
drugs.
Fluorescence microscopy. Latrunculin B was applied to E60K-en-
hanced green fluorescent protein (EGFP) and 34-kDaEGFP Dictyoste-
lium cells 16 to 20 h after induction of E60K expression. The cells were
dislodged, replated on a coverslip, and allowed to adhere for 30 min at
20C. Stock solutions of either 1 mM latB in 20% dimethyl sulfoxide
(DMSO) and Sorensens phosphate buffer, pH 6.1, or 20% DMSO and
Sorensens phosphate buffer were added and allowed to mix by diffusion
to achieve a final concentration of latrunculin B of 10 M or 0 M,
respectively. After incubation for 2 h at 20C, the cells were fixed and
stained with tetramethyl rhodamine isocyanate (TRITC)-phalloidin as
described previously (21). Coverslips were examined on a Zeiss LSM 510
VIS/META confocal microscope. For quantitative fluorescence compari-
sons, the red-channel gain settings for E60K-EGFP and 34-kDaEGFP
Dictyostelium cells were set using the non-latB-treated controls for each
and held constant for recording of latB-treated cells. Quantitative analysis
of changes in TRITC fluorescence was performed using ImageJ (35). Ex-
periments were performed three times.
Study of the size distribution of Hirano bodies was performed by flu-
orescence microscopy. Samples of cells expressing E60K-GFP were fixed
for 25 min in 3.7% formaldehyde in Sorensens buffer with 1 mM EGTA,
coverslips were allowed to air dry and mounted with Crystal Mount
(Biomeda) on microscope slides. Images of the GFP signal were collected
with a Zeiss LSM 510 VIS/META confocal microscope using a 40 ob-
jective. The exposure and illumination settings were kept constant for all
Eukaryotic Cell

FIG 1 Cosedimentation of 34-kDa and E60K proteins with F-actin. Binding
of 3 M E60K or 34-kDa protein as a function of the actin concentration at low
calcium. The E60K protein () saturated at a 2:1 molar ratio (actin/E60K
protein), while the 34-kDa protein () never achieved saturation, even at a
20:1 molar ratio (actin/34-kDa protein). The error bars represent standard
deviations.
the images collected. For all samples, fields of cells were selected ran-
domly, all images were analyzed, and at least 100 Hirano bodies were
counted per condition in each experiment. Each experiment was repeated
at least three times. Hirano body sizes were quantified with ImageJ. Dis-
tributions of Hirano body size were not Gaussian. The effect of drugs on
Hirano body size was assessed by dividing the range of Hirano body sizes
into quintiles. The number of Hirano bodies in each size range in the
drug-treated cells was compared to the control, and the results were ana-
lyzed using the chi-square test.
Transmission electron microscopy. Transmission electron micros-
copy (TEM) was used to determine the structures of Hirano bodies
formed by E60K protein expressed in Dictyostelium. Dictyostelium cells
were prepared for TEM as previously described (36). Cells were embedded
in EMbed 812 (Electron Microscopy Sciences), sectioned using either an
RMC 5000 or MT-X ultramicrotome (RMC Products, Boeckeler Instru-
ments, Inc.) with a diamond knife, and stained with uranyl acetate and
lead citrate. Specimens were examined by transmission electron micros-
copy using a JEOL 1200 EX equipped with a Gatan detector.
F-actin solutions were negatively stained with 2% uranyl acetate and
visualized using a JEOL 100 CXII transmission electron microscope op-
erating at 80 kV as previously described (24). G-actin (10 M) was po-
lymerized in 20 mM PIPES, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM
ATP, 5 mM EGTA, 0.2 mM DTT at 4C overnight and added to either
34-kDa protein or E60K protein to give final concentrations of 5 M and
2.5 M, respectively.
RESULTS
Previous studies have demonstrated that gain-of-function mutant
forms of the 34-kDa protein can induce the formation of model
Hirano bodies when expressed in either Dictyostelium or mamma-
lian cells (1618). During the construction and characterization of
a vector employing the discoidin promoter to drive inducible ex-
pression of the 34-kDa protein fused to GFP, a clone was identified
that showed large aggregates of GFP that colocalized with rhod-
amine-phalloidin staining and appeared similar to model Hirano
bodies. Since model Hirano bodies are not induced by expression
of the wild-type 34-kDa protein alone or fused to GFP (25), we
isolated and sequenced the plasmid from these cells and compared
May 2014 Volume 13 Number 5
Requirements for Hirano Body Formation
the sequence to that expected for 34-kDa proteinGFP. The re-
sults showed a single base change altering codon 60 from GAA,
encoding glutamate, to AAA, encoding lysine (data not shown).
Model Hirano body formation following transformation of cells
with plasmids carrying this sequence was reproducible. The E60K
mutation is in the putative inhibitory region of the 34-kDa pro-
tein, which spans residues 1 to 76 (24). The formation of model
Hirano bodies by mutant 34-kDa proteins is believed to be the
result of increased F-actin affinity and loss of calcium sensitivity
(16, 17). We characterized the mutated E60K protein to further
dissect the properties required for model Hirano body formation.
Biochemical characterization of E60K protein. For biochem-
ical characterization of the E60K protein, the protein was ex-
pressed and purified from Escherichia coli using methods devel-
oped for the wild-type protein (30). As assessed by gel filtration
chromatography, the purified E60K protein was monomeric in
solution, like the wild-type 34-kDa protein (reference 20 and data
not shown). F-actin cosedimentation assays were utilized to char-
acterize the binding of the E60K protein to actin filaments at a low
calcium concentration in mixtures of 3 M 34-kDa or E60K pro-
tein and 3 to 60 M F-actin (Fig. 1). At an equimolar actinto
34-kDa protein concentration, approximately 1 mol of 34-kDa
protein bound to every 5 mol of actin. In contrast, at the same
molar ratio, E60K protein bound to F-actin at 0.65 to 1. Increasing
the total actin concentration to 6 M resulted in nearly all of the
E60K protein being bound (2.62 M) but had only a modest effect
on the total amount of 34-kDa protein bound (0.76 M). Even at
very high actinto34-kDa protein concentrations (20:1), the
binding of the 34-kDa protein did not attain saturation. These
results confirm a substantially higher affinity and maximal level of
binding of E60K protein to F-actin compared to wild-type 34-
kDa protein. To assess the calcium sensitivity, 3 M E60K or
34-kDa protein was mixed with 3 M F-actin at a low or high
calcium concentration. More of the E60K protein bound to
F-actin at both low and high calcium concentrations, as shown
in Table 1. The binding of the E60K and 34-kDa proteins to
F-actin in the presence of a high calcium concentration was
slightly more than half that observed with low calcium in both
cases. Thus, the E60K protein exhibits activated F-actin bind-
ing, but unlike other modified 34-kDa proteins with activated
actin binding that induce formation of Hirano bodies, the
E60K protein retains calcium sensitivity.
Stabilization of actin filaments to latrunculin B-induced de-
polymerization in vitro. Full-length and truncated forms of the
34-kDa protein have been previously shown to inhibit F-actin
depolymerization in a concentration-dependent manner (23, 37).
TABLE 1 Determination of calcium-sensitive binding of 34-kDa and
E60K proteins to F-actina
Concn (M) bound to F-actin
Calcium
Protein Low Ca2 High Ca2 sensitivity (%)b
34-kDa 0.74 0.15 0.44 0.12 59
E60K 1.74 0.26 0.88 0.24 51
a
Solutions of 3 M F-actin and 3 M 34-kDa or E60K protein were cosedimented
under low- or high-calcium conditions (free calcium ion concentrations of 1.4 108
M or 3.1 M, respectively). The E60K protein had calcium-sensitive F-actin binding
similar to that of the 34-kDa protein. More of the E60k protein was bound to F-actin
under high- and low-calcium conditions (P 0.001).
b
Sensitivity (high Ca2/low Ca2) 100.
ec.asm.org 627
Grifn et al.

TABLE 2 Concentrations of F-actin sedimented in the presence of either 34-kDa or E60K protein and of 4.5 M latB for 2 or 24 h
Concn of F-actin (M)
Time in presence latB (% 34-kDa 34-kDa latB (% E60K E60K latB (%
of latB (h) latB depolymerized) latB depolymerized) latB depolymerized)
2 2.36 0.21 0.53 0.24 (78) 2.38 0.11 1.53 0.06 (36) 2.52 0.10 2.60 0.22 (0)
24 2.32 0.11 0.27 0.14 (88) 1.63 0.06 (31) 2.48 0.18 (2)

The 34-kDa protein affects the off rate of actin monomers from
the ends of actin filaments but does not cap the ends, since the
critical concentration is not altered (37). The observation that
the E60K protein had an increased affinity for F-actin compared to
the wild-type 34-kDa protein led us to determine the effect on
F-actin depolymerization. F-actin was effectively depolymerized
in the presence of latB, as shown in Table 2. The presence of the
34-kDa protein partially inhibited latB-induced depolymerization
in vitro. In contrast, the E60K protein completely inhibited depo-
lymerization with no significant change in the F-actin concentra-
tion after either 2 or 24 h in the presence of latB. Thus, the higher
affinity of E60K for F-actin correlates with the ability to inhibit
depolymerization by effectively decreasing the off rate of the actin
monomer from the filament.
E60K protein cross-links and bundles F-actin. The 34-kDa
protein cross-links and bundles F-actin (20, 21). The ability of
E60K protein to cross-link and bundle actin filaments was exam-
ined by negative-stain transmission electron microscopy. Both the
34-kDa protein and the E60K protein cross-linked and bundled
F-actin (Fig. 2). The bundles of actin cross-linked by E60K re-
vealed more cross-striations than those formed with the wild-type
34-kDa protein, likely due to higher ordering and occupancy of
cross-link sites reflecting the higher stoichiometry of binding.
E60K protein expression leads to Hirano body formation.
The observation that E60K protein cross-links and bundles F-ac-
tin together with the gain-of-function F-actin binding was consis-
tent with other mutant forms of the 34-kDa protein that formed
model Hirano bodies. The E60K protein was tested for the ability

FIG 2 Transmission electron micrographs of solutions of F-actin and either the 34-kDa protein or the E60K protein. (A) F-actin. (B and D) F-actin and 34-kDa
protein. (C and E) F-actin and E60K protein. The E60K protein bundles F-actin similarly to the actin-bundling property of the wild-type 34-kDa protein and
appears to show more cross-striations. Scale bars 250 nm (A, B, and C) and 100 nm (D and E).

628 ec.asm.org Eukaryotic Cell


FIG 3 Expression of E60K-GFP protein in abpB Dictyostelium cells leads to
the formation of model Hirano bodies. Dictyostelium cells were transformed
with plasmid to express the E60K protein or the 34-kDa protein fused to GFP.
Cells were examined by differential interference contrast (DIC) microscopy
(left) and by fluorescence microscopy using either GFP (middle) or TRITC-
phalloidin (right). (A) Control cells expressing 34-kDaEGFP do not contain
Hirano bodies. (B) In contrast, E60K-EGFP-expressing cells contain large Hi-
rano bodies. Scale bar 10 m.
to form model Hirano bodies in Dictyostelium lacking the wild-
type 34-kDa protein (abpB).
E60K-EGFP localized in large punctate structures located
within the cytoplasm (Fig. 3). The colocalization of E60K-EGFP
and F-actin within the structures is consistent with the presence of
model Hirano bodies seen in previous studies done with other
mutant forms of the 34-kDa protein (16, 17). Hirano bodies are
defined by their hallmark ultrastructural appearance, which is ob-
served both in Hirano bodies in the brain and in model Hirano
bodies in cell cultures (5, 1618, 38). F-actin filaments are ar-
ranged into a paracrystalline structure that appears as a filament
array with a center-to-center filament spacing of 10 to 12 nm or as
a herringbone structure, depending on the plane of the section.
Transmission electron microscopy was used to examine Dictyoste-
lium cells expressing E60K-EGFP, as shown in Fig. 4. Large inclu-
sions with the hallmark characteristics of F-actin within Hirano
bodies are observed. The E60K protein with the gain-of-function
F-actin binding causes the formation of model Hirano bodies.
This result is consistent with other 34-kDa protein gain-of-func-
tion mutants that induce formation of model Hirano bodies.
Hirano bodies are resistant to latrunculin B drug treatment.
It is known that latB treatment of Dictyostelium induces the depo-
lymerization of the actin cytoskeleton, and latB has been used in
numerous studies to examine the role of actin in such cell pro-
cesses as endocytosis, cell motility, and regulation of contractile
vacuoles (3941). Since the E60K protein reduced F-actin depo-
lymerization in vitro by latB, and since Hirano bodies are com-
posed primarily of actin filaments (5, 7), we used latB to compare
the stability of actin filaments in model Hirano bodies to the other
actin filaments in the cell. The 34-kDaEGFP cells with no latB
treatment had normal cortical 34-kDa protein localization and
colocalization with F-actin, as detected by TRITC-phalloidin
staining (Fig. 5A and B). The E60K-EGFP cells without latB con-
tained large model Hirano bodies that were enriched in E60K-
EGFP and F-actin (Fig. 5E and F). latB treatment of 34-kDaEGFP
cells caused a 77% decrease in F-actin content, as assessed by the
decrease in TRITC-phalloidin staining (Fig. 5D and Table 3). The
34-kDaEGFP fluorescence levels remained high, but the cellular
distribution of the 34-kDa protein appeared to be diffusely dis-
tributed in the cytoplasm, indicating that it was no longer local-
ized in the F-actin cytoskeleton (Fig. 5C). In contrast, E60K-EGFP
cells in the presence of latB had E60K-EGFP localization similar to
that in the absence of latB, as shown in Fig. 5E and G. TRITC-
May 2014 Volume 13 Number 5
Requirements for Hirano Body Formation
FIG 4 Transmission electron micrographs of E60K-EGFP-expressing abpB
Dictyostelium cells with model Hirano bodies. (A) Transmission electron
micrograph of a cell expressing E60K protein with model Hirano bodies. The
model Hirano bodies (HB) are distinct from the electron-dense nuclear ma-
terial (Nuc). Scale bars 1 m (A) and 50 nm (B and C).
phalloidin staining of latB E60K-EGFP cells shows an overall 43%
decrease in cytoplasmic F-actin, significantly less than that ob-
served in latB-treated 34-kDaEGFP cells (Fig. 5F and H and Ta-
ble 3). Measurements of TRITC-phalloidin fluorescence levels
contained within the model Hirano bodies in the presence and
absence of latB showed that only 31% of the F-actin in model
Hirano bodies was susceptible to depolymerization by latB (Table
3). These results show that F-actin within model Hirano bodies is
less susceptible to disassembly by latB, consistent with the hypoth-
esis that increased stability of actin filaments is a property of Hi-
rano bodies that contributes to the formation and assembly of
these structures.
Role of the cytoskeleton in the formation and degradation of
model Hirano bodies. Cytoskeletal structures can contribute to
the formation and degradation of Hirano bodies in a variety of
ways. In order to determine the contributions of the microtubule
and actin cytoskeleton to the formation and degradation of model
Hirano bodies in Dictyostelium, an inducible discoidin promoter
(pVEII) (33) was utilized to drive the expression of E60K-EGFP
and to establish an approximate start time for Hirano body for-
mation. A Dictyostelium autophagy knockout mutant, atg5 (34),
was utilized to slow or delay the degradation of Hirano bodies by
the autophagy pathway (27) and to focus on events during the
formation of Hirano bodies.
Chemical inhibitors were utilized to probe the roles of micro-
tubules and myosin II in model Hirano body formation. Micro-
tubules were depolymerized with nocodazole, the most effective
reagent in Dictyostelium (42). Model Hirano bodies in Dictyoste-
lium are highly enriched for myosin II, but not myosin I (16).
Myosin II function was inhibited with blebbistatin, a specific in-
hibitor of nonmuscle myosin II (43) whose activity has been doc-
umented in Dictyostelium (44). The model Hirano body area was
measured by fluorescence microscopy at 3.5, 5.5, 7.5, and 24 h
after the removal of folate from the media to induce the expression
of the E60K-GFP protein in atg5 cells. Model Hirano bodies were
able to form under all experimental conditions, and all three con-
ditions involved very dispersed size distributions in the Hirano
body area (Fig. 6A to D and Table 4). The efficacy of the drugs was
confirmed by observation of cell rounding and decrease in mem-
ec.asm.org 629
Grifn et al.

turn is smaller than those formed in the presence of blebbistatin.

This is in contrast to the results at 3.5 and 5.5 h. Since the distri-
bution of model Hirano body sizes was highly skewed and differ-
ent for each time and condition, comparison of the size frequency
distribution in the presence of either blebbistatin or nocodazole to
the control was performed by chi-square analysis, as shown in
Table 4. The distribution of model Hirano body sizes formed in
the presence of either nocodazole or blebbistatin showed signifi-
cant differences from the control at different time points, depend-
ing on the drug. These results show that while neither microtu-
bules nor myosin II is required for model Hirano body formation,
they are both required for optimal formation of model Hirano
bodies, and they contribute to different facets of model Hirano
body formation.
Hirano bodies are characterized by hallmark ultrastructural
properties. Electron microscopy was performed at 7.5 h to verify
that model Hirano bodies were formed in the presence of the
drugs in an atg5 background. The paracrystalline order of F-ac-
tin in the model Hirano bodies is shown in Fig. 7. Model Hirano
bodies formed in the presence of either nocodazole or blebbistatin
appeared similar to those formed in the absence of drug (Fig. 7).
Model Hirano bodies formed in the atg5 background also ap-
peared similar in organization to those observed in an AX2 back-
ground. Images were collected at a variety of tilt angles and higher
magnification to gather more information regarding the structure
and organization of the model Hirano bodies (data not shown).
Consistent differences in the structure and order of model Hirano
bodies formed in the presence and absence of nocodazole and
blebbistatin or cell line could not be established.

DISCUSSION
Hirano bodies form under a wide array of stresses and conditions,
but the actual signal or process required for the formation of Hi-
rano bodies is not known. The development of an experimental
system for formation of model Hirano bodies has provided a facile
FIG 5 Hirano bodies are resistant to depolymerization induced by latB. Dic- approach to this question. Three altered forms of the 34-kDa ac-
tyostelium cells expressing either 34-kDaEGFP or E60K-EGFP were incu-
bated with 10 M latB for 2 h and processed for fluorescence microscopy. (A tin-bundling protein induce model Hirano bodies: CT (16), 34-
and B) 34-kDaEGFP-expressing cells have normal cellular localization of kDa EF1 (17), and E60K (present work). CT is a truncated pro-
34-kDa protein (A) and actin cytoskeleton stained with TRITC-phalloidin (B). tein comprising amino acids 124 to 295 (23), while the 34-kDa
(C and D) latB treatment results in redistribution of 34-kDa protein (C) cou- EF1 has a mutation in the first putative EF hand. The E60K
pled to the depolymerization of the actin cytoskeleton, as displayed by loss of
TRITC-phalloidin staining (D). (E) E60K-EGFP-expressing Dictyostelium
protein has a point mutation in codon 60, which is in the region of
cells with model Hirano bodies have E60K protein localized to cellular loca- the inhibitory domain (amino acids 1 to 76), but not in the first
tions consistent with 34-kDa protein but also have E60K protein enrichment interaction zone (amino acids 71 to 123) (24). All three proteins
within the model Hirano body. (F) Corresponding F-actin cytoskeleton and show activated actin binding and cross-linking. The CT and 34-
model Hirano body. (G and H) Incubation of E60K-EGFP model Hirano
body-containing cells with latB results in a modest change in E60K protein
localization (G) and depolymerization of the actin cytoskeleton (H). F-actin
contained within the Hirano bodies appears to be resistant to depolymeriza- TABLE 3 Average mean intensities of TRITC-phalloidin fluorescence
tion by latB, as evidenced by the high intensity of TRITC-phalloidin staining (F-actin) measured in E60K-EGFP- and 34-kDaEGFP-expressing cells
that persists within the Hirano bodies. Scale bar 5 m. in the presence and absence of latrunculin B
TRITC fluorescence % F-actin
(mean intensity decrease with
brane ruffling and movement in the presence of blebbistatin and Cell [relative units] SD) 10 M latB
by staining for microtubules in the presence of nocodazole (not 34-kDaEGFP 45.3 14.3
shown). 34-kDaEGFP latB 10.4 2.4 77
The mean size of model Hirano bodies formed in the presence E60K-EGFP (no Hirano body) 34.5 9.2
of blebbistatin is smaller than those obtained in the presence of E60K-EGFP (no Hirano body) latB 6.4 2.0 81
nocodazole, which is smaller than the control at 3.5 and 5.5 h after E60K-EGFP (Hirano body) 39.4 5.5
induction (Table 4). At 7.5 h, the mean size is not very different. E60K-EGFP (Hirano body) latB 22.3 8.8 43
After 24 h of induction, the mean model Hirano body size is Hirano body only 89.2 0.8
Hirano body only latB 57.2 17.2 31
smaller in the presence of nocodazole than the control, which in

630 ec.asm.org Eukaryotic Cell

 Requirements for Hirano Body Formation

3.5 hour 7.5 hr

35.00 25.00
blebbistatin blebbistatin

30.00
A. nocodazole
control C. nocodazole
control

20.00

25.00

15.00
20.00

%Frequency
%Frequency

15.00
10.00

10.00

5.00
5.00

0.00 0.00
0 100 200 300 400 500 600 700 >800 0 100 200 300 400 500 600 700 >800
Hirano Body Area (pixels)
Hirano Body area (pixels)

5.5 hr 24 hr
25.00 16.00
blebbistatin blebbistatin

B. nocodazole
control 14.00
D. nocodazole
control

20.00
12.00

10.00
%Frequency
15.00
% Frequency

8.00

10.00
6.00

4.00
5.00

2.00

0.00 0.00
0 100 200 300 400 500 600 700 >800
0 100 200 300 400 500 600 700 >800
Hirano Body Area (pixels) Hirano Body Area (pixels)

FIG 6 Representative size distribution of model Hirano bodies as a function of time (shown at the upper left of each panel) in the presence of either nocodazole
or blebbistatin or in the absence of the inhibitors (control). Induction of E60K-GFP expression by the removal of folate is designated time zero. Nascent and
mature model Hirano bodies had diameters of approximately 0.2 m and 2 to 3 m, respectively.

kDa EF1 proteins lack calcium regulation of actin binding, in
addition to possessing activated actin binding. The 34-kDa pro-
tein is regulated by calcium and can bind to the sides of actin
filaments, cross-link actin filaments into bundles, and slow the
disassembly of actin filaments (20, 21, 37). The 34-kDa protein is
localized to transient cellular structures, such as filopodia (21) and
the phagocytic cup (45), as well as the leading and trailing edges of
migrating cells (25). The E60K protein has an enhanced ability to
bind filaments (Fig. 1), to cross-link actin filaments into highly
ordered bundles (Fig. 2), and to slow the disassembly of actin
filaments (Table 2). The binding of the E60K protein to F-actin is
calcium regulated, as is the parental 34-kDa protein (Table 1).
However, this property is not translated into facile disassembly of
the model Hirano body, unlike the transient cellular structures the
34-kDa protein participates in. Probing the stability of actin fila-
ments in the presence of latrunculin B shows that the highly or-
dered actin in model Hirano bodies is more resistant to disassem-
bly than actin filaments in other regions of the cytoskeleton
(Fig. 5).
Previous work in Dictyostelium has shown alternative methods
for the formation of actin aggregates. Jasplakinolide, a toxin de-
rived from a marine sponge, induces formation of actin-rich in-

TABLE 4 Mean sizes of model Hirano bodies formed in the absence or presence of either 10 M nocodazole or 100 M blebbistatin
Hirano body size (mean SD)a P valueb
Time after Blebbistatin vs. Nocodazole vs.
induction (h) Control Blebbistatin Nocodazole control control
3.5 233.5 247.4 144.1 181.6 167.9 194.8 0.01 0.23
5.5 203.2 233.0 149.3 166.8 176.7 146.8 0.01 0.01
7.5 171.9 214.7 162.5 169.4 185.5 220.8 0.72 0.64
24 211.4 182.0 284.2 277.6 147.0 132.3 0.01 0.02
a
Sizes are given in pixels.
b
P values are based on distributing the Hirano body areas into quintiles (chi-square analysis of sample versus control).

May 2014 Volume 13 Number 5 ec.asm.org 631

Grifn et al.

FIG 7 Transmission electron micrographs of E60K-EGFP-expressing atg5 Dictyostelium cells with model Hirano bodies. The cells were fixed for electron
microscopy 7.5 h after induction of E60K expression. (A to F) E60K expressed in wild-type AX2 cells. (G, H, and I) E60K expressed in atg5 cells. (A, D, and G)
No-drug controls. (B, E, and H) Cells treated with nocodazole. (C, F, and I) Cells treated with blebbistatin. The asterisks indicate the locations of the model
Hirano body. Note that panel H has portions of the Hirano body in both longitudinal section and cross section. Model Hirano bodies formed in the presence of
drugs appear similar to those formed in the absence of drugs. In addition, model Hirano bodies formed in an atg5 background are similar to those in the AX2
wild-type background. Scale bars 2 m (top row) and 1 m (middle and bottom rows).

clusions, actin aggresomes, in Dictyostelium and in mammalian
cells (4648). The actin aggresomes are also more resistant to dis-
assembly but lack the paracrystalline order that is characteristic of
Hirano bodies (47). Jasplakinolide stabilizes actin filaments, de-
creases the rate of depolymerization and the critical concentration
of actin, and promotes nucleation of actin filaments (49, 50).
However, jasplakinolide lacks cross-linking activity, suggesting
that cross-linking of actin filaments is an essential element in the
formation of paracrystalline order in Hirano bodies. Overexpres-
sion of VASP (vasodilator-stimulated phosphoprotein) tethered
to endosomes in Dictyostelium produced large actin aggregates
(51). VASP contains 1 actin-binding domain but forms tetramers
in solution and can cross-link actin filaments. Tethering to the
membrane brings VASP and actin filaments into close spatial
proximity. The large actin aggregates did not exhibit the paracrys-
talline order of Hirano bodies, which further suggests that not
only cross-linking, but also bundling of actin filaments, plays a
role in the formation of ordered actin aggregates.
The finding that the E60K protein retains calcium regulation of
actin binding in addition to activation of actin binding (Fig. 1) is
noteworthy, since this result shows that activation of actin binding is
sufficient to trigger formation of Hirano bodies and that loss of cal-
cium regulation is not an additional factor. Taken together, these
results indicate that both stabilization of actin filaments by inhibition
of disassembly and high-affinity cross-linking are required to induce
the formation and maintenance of Hirano bodies in cells.
In the initial stages of model Hirano body formation, stabili-
zation of filaments, alignment, and bundling promote the forma-
tion of small clusters of aggregated filaments. We tested for a pos-
sible role for the cytoskeleton in the formation of Hirano bodies
using the drugs nocodazole and blebbistatin to depolymerize mi-
crotubules and to inhibit myosin II, respectively. A role for micro-
tubules in the formation of model Hirano bodies was suggested by
the initial reports that aggresomes form by transport of small nas-
cent structures to form a single large aggregate in each cell (52
54). Similarly, small nascent structures are transported on micro-
tubule tracks to form actin aggresomes following application of
jasplakinolide (47). Our studies show that model Hirano bodies
form in cells treated with nocodazole but are smaller than in con-
trols. These results indicate that microtubules contribute to the
formation of small aggregates and their growth into large model
Hirano bodies but that microtubules are not required for model
Hirano body formation (Fig. 6 and Table 4).
Our results also support a role for myosin II in the dynamics of

632 ec.asm.org Eukaryotic Cell


early model Hirano body formation, since the size of model
Hirano bodies formed in the presence of blebbistatin grew more
slowly than that of the control (Fig. 6). Blebbistatin is a specific
small-molecule inhibitor of myosin II (43). Blebbistatin binds to
the ADP/Pi state of myosin II and blocks the actin-activated
ATPase of myosin II. Blebbistatin-inhibited myosin can bind actin
but is in a low-affinity conformation (55). In Dictyostelium, bleb-
bistatin blocks myosin II-dependent processes, but not other pro-
cesses known to be dependent on myosins I, V, and VII (44). The
finding that model Hirano bodies formed in the presence of bleb-
bistatin are smaller than controls is consistent with the observa-
tion that model Hirano bodies contain myosin II, but not myosin
I (16). However, these results do not allow us to define the precise
role of myosin II during the formation of Hirano bodies. The
simplest and most direct mechanism would be inhibition of my-
osin II-dependent transport of the nascent model Hirano body
structures into large aggregates. However, myosin II that is
ATPase defective can still provide a function by cross-linking actin
filaments and plays a structural role independent of its motor
function (56). An elegant recent study revealed that nonmuscle
myosin II plays a role in cytokinesis that involves cross-linking
and tension, but not translocation of actin filaments (57). Thus,
myosin II could play a role in the structure and collection of the
actin filaments into the aggregates and/or a role in the transport of
small structures into a single large aggregate in each cell. Taken
together, these findings suggest that myosin II is not required for
Hirano body formation but facilitates the process of forming actin
aggregates and their subsequent coalescence.
An additional possible explanation of the effect of blebbistatin
on the size of model Hirano bodies involves a role of the actin
cytoskeleton and myosin II in degradation of Hirano bodies. The
turnover of model Hirano bodies can occur by autophagy, as well
as the proteasome or exocytosis (27). While the cytoskeleton is not
required for nonselective macroautophagy, the actin cytoskeleton
is needed for selective autophagy in yeast and contributes to atg9
cycling and the formation of the preautophagosomal structure
(PAS) (58). A recent study showed that myosin II is activated by
the kinase atg1, which phosphorylates a myosin light-chain ki-
nase-like protein and contributes to membrane trafficking for the
formation of autophagosomes (59). In this scenario, the smaller
size of the model Hirano body-like structures would involve a
failure in formation and maturation of autophagosomes. Consis-
tent with this possibility is the observation that many of the model
Hirano body structures observed were contained in vesicles in
both the presence and absence of blebbistatin and in both wild-
type cells and cells lacking atg5 (27). The delivery of model Hi-
rano bodies to a vesicular compartment could occur by an alter-
native pathway that is independent of atg5 and atg7 (60). Further
elucidation of the role of myosin II in the dynamics of Hirano
bodies will be an exciting challenge for future investigations.
ACKNOWLEDGMENTS
We thank Dong Huan Kim for initial identification of the E60K mutation.
This work was supported by an NIH award (1R01-NS04645101) to
R.F. and M.F.
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