Molecular Biology of the Cell
Vol. 21, 2707–2720, August 1, 2010
Regulators of the Actin Cytoskeleton Mediate Lethality in
a Caenorhabditis elegans dhc-1 Mutant
Aleksandra J. Gil-Krzewska, Erica Farber,* Edgar A. Buttner,†and Craig P. Hunter
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
Submitted July 21, 2009; Revised May 11, 2010; Accepted June 3, 2010
Monitoring Editor: Erika Holzbaur
Functional analysis of cytoplasmic dynein in Caenorhabditis elegans has revealed a wide range of cellular functions for
this minus-end–directed motor protein. Dynein transports a variety of cargos to diverse cellular locations, and thus cargo
selection and destination are likely regulated by accessory proteins. The microtubule-associated proteins LIS-1 and
dynein interact, but the nature of this interaction remains poorly understood. Here we show that both LIS-1 and the
dynein heavy-chain DHC-1 are required for integrity of the actin cytoskeleton in C. elegans. Although both dhc-1(or195ts)
and lis-1 loss-of-function disrupt the actin cytoskeleton and produce embryonic lethality, a double mutant suppresses
these defects. A targeted RNA interference screen revealed that knockdown of other actin regulators, including actin-
capping protein genes and prefoldin subunit genes, suppresses dhc-1(or195ts)–induced lethality. We propose that release
or relocation of the mutant dynein complex mediates this suppression of dhc-1(or195ts)--induced phenotypes. These
results reveal an unexpected direct or indirect interaction between the actin cytoskeleton and dynein activity.
Cytoplasmic dynein is a minus-end–directed microtubule
motor protein that transports a wide range of cargos, includ-
ing vesicles, organelles, and mRNAs (Mallik and Gross,
2004; Vallee et al., 2004). Dynein is also required for nuclear
and cell migration and to reorient microtubules with respect
to cellular architecture. Dynein is a large multisubunit motor
protein that interacts with a large number of accessory pro-
teins, including the multisubunit dynactin complex, and an
assortment of dynactin and microtubule-binding proteins, to
regulate its diverse interactions and functions (Vallee et al.,
2004). In addition, in order to efficiently transport cargo to
the minus end of microtubules, dynein must relocalize to a
region near the plus end of microtubules. How this large
and growing list of interacting proteins work together to
regulate the spatial and temporal diversity of dynein func-
tion is not understood.
Cytoplasmic dynein is composed of two heavy chains (520
kDa) that contain ATPase and motor activities, two interme-
diate chains (74 kDa) thought to anchor dynein to its cargo,
two light intermediate chains (53–59 kDa), and several light
chains whose functions are poorly understood. The dynein
heavy chain (DHC) is a member of the AAA? (ATPases
associated with diverse cellular activities) family of proteins.
The C-terminus contains six AAA? subunits (AAA1–
AAA6) organized into a ring (Neuwald et al., 1999). The first
four AAA modules (AAA1–AAA4) bind ATP, but only ATP
hydrolysis by AAA1 has been linked to motor activity. A
coiled-coil “stalk” responsible for microtubule binding is
situated between domains AAA4 and AAA5 (Gee et al.,
1997). The N-terminal “stem” region mediates interactions
with the other heavy chain within the dynein complex and
interactions with intermediate and light chains as well as the
dynactin complex. Each of these dynein subunits and the
multisubunit dynactin complex have been implicated in
cargo binding and regulation of dynein activity (Gibbons et
al., 1991; Ogawa, 1991; Koonce et al., 1992).
Cytoplasmic dynein function and localization has been
extensively studied in the early Caenorhabditis elegans em-
bryo. DHC localization by immunofluorescence shows dy-
namic cell cycle–dependent patterns in C. elegans embryos
(Gonczy et al., 1999; Schmidt et al., 2005). DHC is distributed
throughout the cytoplasm but is enriched at the nuclear
envelope during prometaphase, at the spindle midzone dur-
ing metaphase, and at the cell cortex in two-cell stage em-
bryos. Small pools of dynein were also observed on the
entire metaphase spindle. Dynein function in embryos has
been investigated using RNA interference (RNAi) and con-
ditional mutant alleles (Gonczy et al., 1999; Yoder and Han,
2001; Cockell et al., 2004). One of these conditional alleles,
dhc-1(or195ts), is a mis-sense mutation substituting serine for
leucine at amino acid 3200 (S3200L) in the DHC microtu-
bule-binding stalk region. At the permissive temperature
(15°C), dhc-1(or195ts) provides sufficient dynein activity for
normal development, but growth at the nonpermissive tem-
perature (25°C) results in embryonic lethality and adult
sterility. The phenotype of dhc-1(or195ts) animals and their
progeny grown continuously at the permissive temperature
is similar to that of the wild type. However, at the nonpermis-
sive temperature the dhc-1(or195ts) phenotype is indistinguish-
able from that of the strong loss-of-function phenotype pro-
duced by dhc-1 RNAi. Shifting these temperature-sensitive
mutant animals and embryos to the nonpermissive temper-
ature rapidly disrupts or reduces dynein function, revealing
a wide range of defects, indicating that dynein is required
for many microtubule-dependent processes, including pro-
This article was published online ahead of print in MBoC in Press
on June 16, 2010.
Present address: * Jules Stein Eye Institute, UCLA, 100 Stein Plaza,
Los Angeles, CA 90095;
School, Department of Psychiatry, Mailman Research Center, Bel-
mont, MA 02478.
Address correspondence to: Craig Hunter (email@example.com.
†McLean Hospital/Harvard Medical
© 2010 by The American Society for Cell Biology2707
nuclear migration, spindle assembly, positioning and orien-
tation, chromosome segregation, and cytokinesis (Gonczy et
al., 1999; Yoder and Han, 2001; Schmidt et al., 2005). DHC-
1(S3200L) localization at the nonpermissive temperature
shows accumulation near the minus ends of centrosomal
microtubules (Schmidt et al., 2005).
A variety of accessory proteins, including the dynactin
complex and the microtubule-binding protein LIS-1, regu-
late cargo selection and dynein activity. The multisubunit
dynactin complex attaches dynein to kinetochores and ve-
sicular organelles and in vitro functions to increase dynein
processivity (King, 2000). LIS-1, which acts to increase dy-
nein ATPase activity, binds to two sites on dynein: the first
site is located in the region also responsible for binding site
of the intermediate chains involved in cargo binding, and
the second binding site is the P-loop involved in motor
activity (Sasaki et al., 2000; Tai et al., 2002; Mesngon et al.,
2006). Coimmunoprecipitation studies show that LIS-1 and
dynein can interact in vivo (Faulkner et al., 2000; Smith et al.,
2000), but the character of this interaction remains elusive.
LIS-1 also interacts with the dynamitin subunit of dynactin
(Karki and Holzbaur, 1999; Tai et al., 2002). Because LIS-1
interacts with the dynein–dynactin complex, LIS-1 was
thought to be a structural part of the motor complex (Liu et al.,
1999; Faulkner et al., 2000; Smith et al., 2000). However, because
LIS-1 increases dynein ATPase activity, it is more likely to
perform a regulatory function (Mesngon et al., 2006).
In C. elegans, dynein localization is dependent on LIS-1,
and LIS-1 localization, in turn, is dependent on dynein
(Cockell et al., 2004). In the germline LIS-1 is expressed in the
cytoplasm and is enriched at the nuclear envelope of oocytes
(Buttner et al., 2007). In embryos LIS-1 localization is cell
cycle-dependent. During late prophase LIS-1 localizes inside
pronuclei in the one-cell stage embryo and at the nuclear
periphery and around chromosomes in the two-cell stage
embryo. During metaphase and anaphase LIS-1 localizes to
the spindle, and in late anaphase and telophase LIS-1 is
enriched at the ends of microtubules asters. LIS-1 also local-
izes along the microtubules (Cockell et al., 2004). In one-cell
stage embryos, lis-1 loss of function phenocopies dhc-1 loss
of function, causing defects in spindle assembly, pronuclear
migration and centrosome separation (Cockell et al., 2004).
staining of extruded gonads to visualize F-actin (red) and DNA (blue), respectively. (A) A single optical section of the pachytene germline
showing the F-actin structure of the cytoplasmic rachis in wild type (N2), dhc-1(or195ts), lis-1(n3334), and in dhc-1(or195ts); lis-1(RNAi). In
wild-type gonads the germline rachis is straight with regularly positioned nuclei in the surrounding cortex, whereas in both mutants the
F-actin lining the rachis is ruffled, and many cortical nuclei are displaced into the rachis. (B) 3D reconstruction of F-actin serial optical images
to better visualize rachis abnormalities, deformation of the cytoskeleton, and irregularity of actin cages that connect nuclei to the cytoplasmic
rachis (arrows). (C) Defects in cortical nuclear localization. Labeled as in A. Images in the leftmost column represent a single optical section
near the gonad surface. Areas in white squares are magnified and displayed to the right to illustrate the regular actin network surrounding
individual nuclei in wild type and the absence of nuclei or clusters of 2–3 nuclei within an actin ring in both mutants. Distal (D) and proximal
(P) orientations of the gonad is indicated on the merged image. Scale bar, 10 ?m.
Mutations of dhc-1 and lis-1 disrupt F-actin organization in the pachytene region of the gonad. Phalloidin-rhodamine and Hoechst
A. J. Gil-Krzewska et al.
Molecular Biology of the Cell2708
pressing the embryonic lethality. These observations may
explain both the allele specificity of the cosuppression and
the wide variety of functions that can be disrupted to restore
dhc-1 function (see below).
CAP-1 and -2 form heterodimers of actin-capping protein,
and RNAi knockdown of each was nearly indistinguishable.
Capping proteins bind the barbed end of actin with high
affinity, inhibiting both assembly and disassembly of actin
monomers (Wear and Cooper, 2004). In the gonad and at
cell–cell junctions in early embryos, depletion of capping
proteins caused an increase in F-actin levels, indicating that
capping proteins were acting to limit actin assembly in these
locations. Thus, the increase in DHC-1 localization to normal
sites in response to increased F-actin content may represent
either recruitment or redistribution of dynein. However
treatment with the actin polymerization promoting peptide
jasplakinolide caused an increase in F-actin levels and a
redistribution of DHC-1, suggesting that F-actin levels and
not an unknown effect of capping protein activity drive the
redistribution of DHC-1.
Our screen revealed that the capping proteins, CAP-1 and
-2, as well as the dynein light IC,*** DLI-1, suppress dhc-1
lethality, although not as strongly as lis-1 knockdown. In
addition to these four moderately strong suppressors, we
identified 32 additional suppressors that we have not further
characterized. Interestingly, half of these are known or sus-
pected to regulate actin or microtubule dynamics or func-
tion. These results supplement and expand those of
O’Rourke et al. (2007), who performed a genome-wide RNAi
screen for suppressors of dhc-1(or195ts)-induced lethality.
None of the genes identified here were found in the screen
by O’Rourke et al. (2007). This discrepancy might be due to
different testing conditions. Our screen selected for fertile
adults by exposing fourth-stage larvae to RNAi treatment at
a completely nonpermissive 25°C growth temperature. In
contrast, O’Rourke et al. (2007) exposed first-stage larvae to
a semipermissive 23°C temperature resulting in substan-
tially longer RNAi exposure covering many more develop-
mental events. O’Rourke et al. (2007) may not have identified
the same genes either because these genes fail to suppress
dhc-1 at the earlier stages of development or because these
genes have essential functions during the extended growth.
We imagine two mechanisms for suppression of dhc-
1(or195ts). First, the DHC-1(or195ts) mutant protein localizes
strongly to microtubules and MTOCs, and our RNAi knock-
downs shift DHC-1(or195ts) to a more wild-type distribu-
tion. Thus, DHC-1(or195ts) may have normal motor func-
tions but may maintain a persistent attachment to the minus
ends of microtubules; perturbations that release DHC-
1(or195ts) from microtubules may allow dynein to reengage
in another round of normal motor function. Because LIS1 is
thought to be a processivity factor (Coquelle et al., 2002;
Mesngon et al., 2006), reducing LIS-1 levels may increase the
release rate of DHC-1(or195ts) from microtubules. Similarly
in Aspergillus nidulans a NUDF/LIS1 deletion mutant is sup-
pressed by a dynein heavy-chain mutation that reduces
ATPase activity and that causes an increase in dynein local-
ization to microtubules (Zhuang et al., 2007). Thus the lack of
a proposed processivity factor is suppressed by a mutation
that increases the interaction between dynein and microtu-
bules. This hypothesis is appealing, as it explains why both
specific and nonspecific treatments restore dynein function.
However, because different treatments had distinct effects
on DHC-1 localization, a second proposed mechanism is
that RNAi knockdowns redistribute DHC-1 by altering dy-
nein targeting. LIS-1 is involved in targeting dynein to mi-
crotubule structures. Overexpression of LIS1 alters dynein
distribution at the cell cortex (Faulkner et al., 2000). By
analogy, disruption of lis-1 expression could impair dynein
targeting, resulting in the observed increase of dynein pool
in the cytoplasm. In contrast, knockdown of capping pro-
teins resulted in strong localization of dynein to areas en-
riched with F-actin, as well as to the perinuclear region.
Although dynein does not interact directly with F-actin, it
could be anchored to cortical F-actin by other proteins, e.g.,
CLIP-170 or dynactin. An increase in the local concentration
of F-actin and, thus, of dynein-anchoring proteins could
explain the accumulation of dynein at sites with enriched
F-actin. Indeed, our experiments with an actin polymeriza-
tion–enhancing drug, jasplakinolide, showed similar effects
to those caused by knockdown of capping proteins. We were
unable to rescue dhc-1(or195ts) lethality by treatment with
jasplakinolide, but this result was not surprising, as suppres-
sion of lethality likely requires both the restoration of local-
ization and proper activity of DHC-1(S3200L).
In this study we found that the heat-sensitive allele of the
dynein heavy chain, dhc-1(or195ts), disrupts the actin cy-
toskeleton in the C. elegans gonad. Surprisingly, depletion of
factors that regulate dynein activity (lis-1and dli-1) and actin
assembly (actin-capping protein and prefoldin) suppressed
not only the temperature-sensitive lethality associated with
this allele, but also restored the integrity of the actin net-
work. Depletion of these factors in a wild-type background
similarly disrupted actin structures, suggesting that despite
causes dynein relocalization in the germline. Anti-actin antibody
(green), anti-DHC-1 (red), and Hoechst staining of gonads (pachytene
region) extruded from wild-type (N2) animals and incubated with
either 0.1% MeOH (vehicle) or 1 ?M jasplakinolide for 30 min at room
temperature before fixation (see Materials and Methods). The pachytene
region of the germline is shown in the top row of each panel (scale bar,
10 ?m), and close-up of nuclei is shown in the bottom row (scale bar,
1 ?m). The jasplakinolide-treated germline exhibits strong perinuclear
of cap-1 and -2 RNAi germlines. Distal (D) and proximal (P) orienta-
tions of the gonad is indicated on the merged image.
Jasplakinolide treatment increases F-actin levels and
Actin Regulators Interact with Dynein
Vol. 21, August 1, 2010 2719
the disparate processes mediated by these factors, a common
mechanism is likely responsible for the suppression of dhc-
1(or195ts) lethality. At the elevated temperature the mutant
DHC-1 protein accumulated near the minus ends of micro-
tubules; the above depletions caused DHC-1 to redistribute
toward a more normal pattern. Therefore, we propose that
the common bypass mechanism is decreased dynein proces-
sivity that allows the mutant DHC-1 protein to release from
near microtubule minus ends and reinitiate a new functional
interaction nearer the plus end of the microtubules. This
work, thus illustrates a previously undescribed and likely
indirect interaction between microtubule and actin cytoskel-
We thank Pierre Go ¨nczy and Susan Strome for generous gifts of antibodies,
Hunter lab members for advice and discussions, and Konrad Krzewski for
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