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 (firstname.lastname@example.org.
†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
Interestingly, in migrating neurons and in Dictiostylium, re-
duction in LIS-1 levels reduces filamentous (F)-actin content
(Kholmanskikh et al., 2003; Rehberg et al., 2005). The reduc-
tion in F-actin is associated with reduced Cdc42 and Rac1
activity and altered actin dynamics. Although these obser-
vations link LIS-1 activity to regulation of the actin cytoskel-
eton, the nature of this interaction is still unknown.
Here we show that although C. elegans lis-1 is an essential
gene, lis-1 knockdown by RNAi in dhc-1(or195ts) animals
suppresses the lethality of both dhc-1(or195ts) and lis-1
RNAi, resulting in viable progeny. To investigate this sur-
prising result, we used a genetic interactions database to
assemble a list of 238 additional candidate DHC-1– and
LIS-1–interacting proteins (Zhong and Sternberg, 2006) and
then screened these genes for those that, like lis-1, when
depleted by RNAi, suppress the temperature-sensitive le-
thality of dhc-1(or195ts). We found four genes, lis-1, cap-1,
cap-2, and dli-1, that when depleted by RNAi significantly
suppressed dhc-1(or195ts) lethality, cytoskeletal defects, and
DHC localization in the germline and early embryo. Two of
these genes, lis-1 and dli-1, are known dynein regulators,
and lis-1, cap-1, and cap-2 are known or implicated regulators
of F-actin dynamics. We propose that the unexpected rescue
of dhc-1(or195ts) defects may be mediated by treatments that
release or relocate the mutant dynein complex from the
minus ends of microtubules.
MATERIALS AND METHODS
Nematode Strains and Culture Conditions
The following mutant strains were used in this study: C. elegans Bristol strain
N2 (wild type), EU828, dhc-1(or195ts) I; MT12272, juIs73 III/lis-1(n3334) III;
n3334 contains a deletion encompassing bases 4325–6342 of cosmid T03F6;
HR10, dhc-1(ct42) dpy-5(e61)/unc-11(e47) bli-4(e937) I; KR332, dhc-1(h79) dpy-
and Hoechst staining of extruded gonads (pachytene region) to visualize microtubules (green), F-actin (red), and nuclei (blue), respectively.
The microtubule cytoskeleton of wild-type (N2) and mutant (dhc-1 and lis-1) gonads from animals cultured at indicated temperatures do not
show any obvious abnormalities, whereas both mutants show obvious defects in the actin cytoskeleton. Distal (D) and proximal (P)
orientations of the gonad is indicated on the merged image. Scale bar, 10 ?m.
Microtubule organization in lis-1 and dhc-1 mutants is comparable to wild type. Anti-?-tubulin antibody, phalloidin-rhodamine,
Actin Regulators Interact with Dynein
Vol. 21, August 1, 20102709
5(e61) unc-13(e450) I; sDp2(I;f). Nematodes were grown under standard cul-
ture conditions at 15–25°C. EU828was maintained at 15°C.
Gene Selection, Screen (RNAi), and Viability Assessment
To create a list of probable lis-1– and dhc-1–interacting genes, we used the
computational data search system “Predictions of C. elegans Genetic Interac-
tions” (http://tenaya.caltech.edu:8000/predict/; Zhong and Sternberg, 2006).
As input genes, we used lis-1 and dhc-1 followed by cap-1 and -2. Candidate
genes (n ? 238) were screened using a genome-wide C. elegans RNAi library
(Geneservice, Cambridge, United Kingdom). The library was constructed by
J. Ahringer’s group at the Wellcome CRC Institute, University of Cambridge,
Cambridge, England. The primary screen was performed on 24-well plates
(CorningCostar, Acton, MA) containing NG medium supplemented with 5
mM IPTG and 100 mg/ml carbenicillin. Plates were seeded with bacteria
cultures and left overnight at room temperature to induce double-strand RNA
(dsRNA) production. The next day two or three dhc-1(or195ts) L4 larvae were
placed in each well, and the plates were incubated at 25°C. Seventy-two hours
later each well was scored for viable progeny. All genes were tested at least
three times. Genes for which knockdown resulted in any number of viable
progeny were tested further.
For quantitative assessment of the suppression of dhc-1(or195ts) lethality,
genes identified in the primary screen were retested on concentrated induced
cultures, prepared as follows. Bacterial cultures were grown overnight and
next diluted 1:3 followed by induction for 4 h at 37°C in LB media containing
1 mM IPTG and 100 mg/ml carbenicillin to produce dsRNA. Five milliliters
of each culture was then concentrated and seeded into each well of a 24-well
plate. Single animals were added to each well and after 24 h at 25° transferred
to a similarly prepared fresh well for an additional 24 h. These plates were
incubated an additional 24 h to allow all viable embryos to hatch. The average
number of viable progeny per animal was calculated for each 24-h period
from at least three separate trials. For experiments in which two genes were
targeted (e.g., cap-1 and -2), the bacterial cultures were concentrated and
mixed in a 1:1 ratio.
To assess the allele specific cosuppression of lis-1 and dhc-1, we grew strain
KR332 on lis-1 RNAi food. Twenty animals produced no viable embryos.
Staining and Microscopy
F-actin and tubulin staining was performed on extruded gonad arms adhered
to poly-l-lysine–coated slides and fixed in 4% formaldehyde in PBS for 40
min at room temperature. The slides were then rinsed in PBS, and the samples
incubated with FITC-conjugated anti-?-tubulin mAb (1:50; Sigma-Aldrich, St.
Louis, MO) overnight at room temperature, followed by incubation with
rhodamine-conjugated phalloidin (0.164 ?M; Invitrogen, Carlsbad, CA) for
2 h. Finally, slides were incubated in 1?x Hoechst (Invitrogen) for 5 min.
Between incubations slides were washed in 1? PBST.
DHC-1 and microtubule staining of one-cell stage embryos was as described in
ine-conjugated AffiniPure anti-rabbit IgG (Jackson ImmunoResearch, West
used at 1:50. Incubation time for all antibodies was 1 h.
dhc-1(or195ts) animals. (A) Average number of
viable hatched larvae laid by dhc-1(or195ts) ani-
mals in two consecutive 24-h periods on control
bacteria or bacteria producing lis-1 dsRNA [lis-
1(RNAi)]. Embryos were incubated an additional
24 h to assess hatching. n ? 3; error bars, ?SEM.
(B) Phalloidin-rhodamine and Hoechst staining
of extruded gonads (pachytene region) to visual-
ize F-actin (red) and DNA (blue), respectively,
from dhc-1(or195ts) animals grown on control or
lis-1 dsRNA-expressing bacteria at the nonper-
missive temperature (25°C). 3D reconstruction of
F-actin serial optical images. Depletion of lis-1 by
RNAi greatly improved F-actin structure, compa-
rable to N2 control (Figure 1A). Distal (D) and
proximal (P) orientations of the gonad is indi-
cated on the merged image. Scale bar, 10 ?m.
RNAi of lis-1 suppresses lethality of
A. J. Gil-Krzewska et al.
Molecular Biology of the Cell2710
F-actin, microtubules, and DHC-1 staining utilized a permeabilization pro-
tocol adapted from Huang et al. (2002) and then fixed in Cytofix solution (BD
Biosciences, San Jose, CA) for 5 min at ?20°C. Slides were first incubated with
anti-DHC-1 polyclonal antibodies (gift from Pierre Gonczy or from Susan
Strome, Indiana University; 1:100) and then rhodamine-conjugated AffiniPure
anti-rabbit IgG (Jackson ImmunoResearch, 1:100). The slides were then incu-
bated with FITC-conjugated anti-?-tubulin mAb (1:50), followed by incuba-
tion with AlexaFluor 405–conjugated phalloidin (Invitrogen).
All images were obtained using an Axiovert 200 spinning disk confocal
microscope (Zeiss, Thornwood, NY) and analyzed with Axiovision software
Jasplakinolide (Invitrogen) was kept as 1 mM solution in anhydrous metha-
nol. To obtain 1 ?M concentration, 1 ?l of stock solution was added into 999
?l of M9. As a control we used M9 with the same volume of anhydrous
methanol. Twenty-four-old adult animals were suspended in M9 medium
containing either jasplakinolide or methanol and transferred onto poly-l-
lysine–coated slides. Gonads were extruded from the animals, and slides
were incubated in humid atmosphere for 30 min at room temperature,
washed once in PBST, fixed with methanol, and then stained with anti-actin
and anti-DHC-1 antibodies.
Densitometric analysis of lane profile plots was performed using ImageJ
(http://rsb.info.nih.gov/ij/), according to guidelines in ImageJ documenta-
tion. The peak areas (corresponding to gel band intensities) of either LIS-1 or
ZK858.3 (CTRL) were measured, and each peak was normalized to the
appropriate peak area of the loading controls for the corresponding amount
of RNA template used.
Loss of Function of Either dhc-1 or lis-1 Disrupts F-Actin
Organization in the Pachytene Region of the Gonad
lis-1 and dhc-1 are required for similar microtubule-depen-
dent cell cycle events in the early C. elegans embryo. In the
one-cell embryo lis-1 and dhc-1 loss-of-function both pro-
duce defects in centrosome separation, pronuclear migra-
tion, and spindle assembly (Cockell et al., 2004). In addition,
lis-1 is required for germline development; lis-1 loss of func-
tion reduces the proliferative capacity of the germline in part
by disrupting the mitotic spindle (Buttner et al., 2007). To
determine whether dhc-1 loss of function causes similar
germline phenotypes, we compared the earliest effects of
lis-1(n3334) and dhc-1(or195ts) on the development of the C.
elegans germline, with a particular emphasis on the structure
of the cytoskeleton.
dhc-1(or195ts) animals (dhc-1) at permissive (15°C) or nonpermissive (25°C) temperatures and fed with either control or lis-1 dsRNA-
producing bacteria [lis-1(RNAi)] were stained with anti-?-tubulin (green), anti-DHC-1 (red), and Hoechst (blue). Individual channels are
shown in grayscale, and merged images are shown in color. To better visualize localization of dynein with respect to microtubules, the last
column (5?) shows magnification of the white square–marked area of merged image; scale bar, 1 ?m. lis-1 knockdown restored dynein
localization to near wild type. Scale bar, 5 ?m.
lis-1 knockdown restores DHC-1 localization in dhc-1(or195ts) embryos. One-cell stage embryos from wild-type animals (N2) or
Actin Regulators Interact with Dynein
Vol. 21, August 1, 2010 2711
We used immunofluorescence microscopy to visualize
and compare F-actin and microtubule cytoskeletal struc-
tures and organization in wild-type, lis-1(n3334), and dhc-
1(or195ts) gonads and germlines (Figure 1). The C. elegans
germline is syncytial, existing as proliferating and differ-
entiating nuclei in a cytoplasmically continuous environ-
ment. The development of these nuclei is organized along
the distal to proximal axis. At the distal end nuclei prolifer-
ate mitotically in a stem-cell-like niche. Proliferating nuclei
exit this niche and begin meiosis in the transition zone,
where chromosome pairs align and the synaptonemal com-
plex begins to form. In the pachytene region, the nuclei are
arranged around the cortex of the gonad creating a nuclei-
free rachis. F-actin staining in wild type showed a cage-
shaped structure surrounding each cortically localized nu-
cleus (Figure 1C). This actin cage opens to the rachis through
uniformly shaped “windows.” F-actin, visualized by three-
dimensional reconstruction from serial optical sections,
showed a straight smooth structure along the edge of the
rachis (Figure 1B). In lis-1(n3334) and dhc-1(or195ts) animals
the structure of F-actin in the pachytene region was dis-
rupted. The structure of the rachis, rather than being straight
and smooth, was ruffled and deformed (Figure 1A). Further-
more, a single actin cage often surrounded multiple nuclei,
and many actin-free nuclei accumulated within the rachis
(Figure 1C). Additionally, the windows connecting nuclei
with the cytoplasm were larger and irregular (Figure 1B).
The differences in the distal gonad were largely restricted
to the pachytene region; few of these defects were evident in
the most distal portion of the gonads (Supplementary Figure
1). These results suggest that both dhc-1 and lis-1 function in
actin cytoskeleton organization. Alternatively, similar effects
could result from the fact that LIS-1 and dynein interact with
each other (Faulkner et al., 2000; Smith et al., 2000), or the
defects in the actin cytoskeleton might be secondary to de-
fects in microtubule organization.
The microtubule cytoskeleton in wild-type animals was
composed of long evenly distributed microtubules within
the gonad rachis, creating a regular network (Figure 2). Each
nucleus on the periphery of the rachis was surrounded by a
regular network of microtubules. In lis-1(n3334) and dhc-
1(or195ts) gonads the microtubule cytoskeleton was similar
to that in wild-type gonads. We noticed some subtle changes
in microtubule organization within the rachis; however,
these changes were most likely caused by defects in the
organization of the actin cytoskeleton surrounding the ra-
chis (Figure 2). Thus, it is unlikely that the common actin
cytoskeleton defects observed in lis-1 and dhc-1 are second-
ary to defects in microtubule organization.
ity affect dynein localization in wild-type em-
bryos differently. Four-cell stage embryos from
wild-type animals (N2) animals fed with either
control bacteria or bacteria expressing the in-
dicated dsRNA were stained with anti-?-tubu-
lin (green), anti-DHC-1 (red), and rhodamine
phalloidin (blue). Individual channels are
shown in grayscale, and merged images are
shown in color. Each image shows a single
optical section of a four-cell stage embryo.
RNAi knockdown of cap-1 and -2 causes
DHC-1 localization to regions of cell-cell con-
tact, an increase in F-actin levels, and the ac-
cumulation of short acting filaments. RNAi
knockdown of dli-1 and lis-1 causes DHC-1
localization to the cytoplasm. Scale bar, 5 ?m.
Suppressors of dhc-1(or195ts) lethal-
A. J. Gil-Krzewska et al.
Molecular Biology of the Cell 2712
The Cytoskeletal Defects and Embryonic Lethality
Associated with dhc-1(or195ts) and lis-1(RNAi) Are
Cosuppressed in dhc-1(or195ts); lis-1(RNAi) Double
The above results indicated that the disruption of actin
structures in lis-1(n3334) and dhc-1(or195ts) was not simply
secondary to defects in microtubule disruption. Therefore, to
determine whether depletion of both lis-1 and dhc-1 would
produce a more severe phenotype and thereby reveal non-
overlapping requirements for actin organization, we used
RNAi to deplete lis-1 in a dhc-1(or195ts) background at the
nonpermissive temperature of 25°C. Surprisingly, codeple-
tion of lis-1 and dhc-1 produced viable embryos. In contrast,
depleting lis-1 in this background at the permissive temper-
ature (15°C; data not shown) or simply maintaining the
dhc-1(or195ts) animals at 25°C produced no viable embryos
(Figure 3A). Because both lis-1(n3334) and dhc-1(or195ts) mu-
tations produced similar dramatic changes in F-actin in the
germline (Figure 1), we asked whether these abnormalities
were present in the double knockdown animals. We found
that the F-actin structures in the double lis-1(RNAi) dhc-
1(or195ts) animals were restored to a level that functioned to
maintain nuclei in the periphery (Figures 1 and 3B, and
Figure S2). Likewise the microtubule cytoskeleton in the
double mutants was comparable to wild type; we did not
observe any changes in the length of microtubules or in
microtubule organization (not shown). Thus, although the
disruption of expression of either lis-1 or dhc-1 alone causes
similar cytoskeletal defects and embryonic lethality, the
codepletion of two genes cosuppress each other for both
The Observed Cosuppression Depends on the
dhc-1(or195ts) Allele and lis-1(RNAi)
The observed cosuppression was between a temperature-
sensitive dhc-1(or195ts) allele that likely disrupts but does
nein localization in the wild-type germline.
Anti-?-tubulin, anti-DHC-1, and Hoechst stain-
ing of wild-type gonads to visualize microtu-
DHC-1 and tubulin localization in the mitotic,
pachytene, and oocyte regions of the gonad. (B)
Perinuclear localization of DHC-1 and tubulin in
the indicated regions of wild-type germline.
Scale bar, 1 ?m. DHC-1 localization is perinu-
clear in the transition zone and pachytene re-
gion. DHC-1 and tubulin do not colocalize. Scale
bar, 10 ?m.
Cytoplasmic and perinuclear dy-
Actin Regulators Interact with Dynein
Vol. 21, August 1, 20102713
not eliminate dynein function and RNAi depletion of lis-1,
which likely substantially reduces, but does not eliminate
LIS-1 activity. Therefore, we determined whether other dhc-
1alleles and null alleles of lis-1 can be similarly suppressed.
We found that the dhc-1(h79) allele was similarly lethal, but
that the lethality was not suppressed by lis-1 RNAi (data not
shown). Similarly, the lis-1(n3334) null allele was not sup-
pressed by dhc-1 RNAi (not shown). Further, we were un-
able to recover a viable dhc-1(or195ts); lis-1(n3334) double
mutant strain. We conclude that the cosuppression is allele-
specific and requires both the dhc-1(or195ts) mutant and
reduced but not eliminated lis-1 function.
lis-1(RNAi) Rescues DHC-1(S3200L) Mislocalization
To investigate the nature of this allele-specific cosuppression
we examined dynein localization in both one-cell embryos
and the germline. As previously reported, at metaphase in
wild-type one-cell embryos cytoplasmic and centrosome-
associated DHC-1 localizes to the spindle on both sides of
the metaphase plate (Gonczy et al., 1999). Although dhc-
1(or195ts) embryos are fully viable at the permissive temper-
ature, the localization of the mutant DHC-1(S3200L) was
altered (Figure 4). In addition to the spindle localization at
metaphase, DHC-1(S3200L) remained detectable both in the
cytoplasm and at the centrosome throughout the cell cycle.
DHC-1(S3200L) was also enriched in the spindle midzone.
Similar to previous reports (Schmidt et al., 2005), at the
nonpermissive temperature DHC-1(S3200L) localized pri-
marily to the minus end of microtubules, close to or at the
microtubule-organizing center (MTOC), which indicates de-
fects in releasing dynein and its relocalization rather than in
movement.(Figure 4). RNAi-mediated knockdown of lis-1 in
(red), and rhodamine phalloidin (blue) staining of wild-type (N2) and the indicated RNAi-treated gonads. Individual channels are shown in
grayscale, and merged images are shown in color. A fragment of the distal gonad is shown in the top row of each panel (scale bar, 5 ?m)
and a close-up of a single nucleus is shown in the bottom row (scale bar, 1 ?m). cap-1 and -2 RNAi cause notable increase in F-actin levels
and striking localization of DHC-1 to the perinuclear region. In contrast lis-1 and dli-1 RNAi cause an increase in cytoplasmic DHC-1
localization. Distal (D) and proximal (P) orientations of the gonad is indicated on the merged image.
Suppressors of dhc-1(or195ts) lethality differently affect dynein localization in the germline. Anti-?-tubulin (green), anti-DHC-1
A. J. Gil-Krzewska et al.
Molecular Biology of the Cell2714
wild-type animals, similar to the lis-1(null), caused DHC-1 to
become cytoplasmic (Figure 5 and Cockell et al., 2004). How-
ever knockdown of lis-1 in dhc-1(or195ts) embryos altered
DHC-1(S3200L) localization, such that a portion of DHC-
1(S3200L) protein was distributed along microtubules (Fig-
ure 4). Compared with wild-type embryos, there was more
dynein localized to microtubules surrounding centrosomes
in the dhc-1(or195ts); lis-1(RNAi) embryos. Furthermore,
while DHC-1(S3200L) was detected on microtubules at the
metaphase plate, the level of staining was generally reduced
compared with wild type. Finally, in some embryos DHC-1
was only localized along microtubules rather than on the
metaphase plate (not shown).
In the germline of wild-type animals, DHC-1 localization
was perinuclear throughout the whole gonad, except in
oocytes where the localization was cytoplasmic (Figure 6). In
the transition zone the staining was more punctuated but
remained perinuclear (Figure 6B). We failed to detect a
significant overlap between dynein localization and micro-
tubules in either wild-type or dhc-1(or195ts) gonads. We also
did not detect any colocalization between dynein and F-
actin. Similar to the observations in embryos, LIS-1 is re-
quired for DHC-1 localization in gonads, as knockdown of
lis-1 caused the perinuclear-localized dynein to become cy-
toplasmic (Figure 7). This result suggests that LIS-1 may
regulate dynein localization or be required for dynein at-
tachment to microtubules.
Disruption of F-Actin Dynamics Suppresses dhc-1(or195ts)
To gain insight into how reduction of lis-1 suppresses
dhc-1(or195ts) embryonic lethality, we sought to identify
additional genes that when depleted by RNAi also sup-
pressed the temperature-sensitive embryonic lethality of
dhc-1(or195ts). We first compiled a list of 238 candidate
DHC-1– and LIS-1– interacting genes using the computa-
tional data search system “Predictions of C. elegans Genetic
Interactions” (Table S1; Zhong and Sternberg, 2006; see Ma-
terials and Methods). We selected genes predicted to interact
with dynein and LIS-1. Available bacterial clones for these
genes were selected from an RNAi bacterial clone library,
and dhc-1(or195ts) L4 animals were grown on these clones as
the sole food source at 25°C. To score for suppression of
dhc-1(or195ts) lethality, we determined the viability (hatch-
ing) of progeny produced in the subsequent 24 h. Bacterial
clones that resulted in any viable dhc-1(or195ts) progeny
were sequenced to verify the identity of the insert and
retested in triplicate for suppression of dhc-1(or195ts). Over-
all, 36 clones produced viable progeny (Table S2). Among
these genes, lis-1 RNAi produced the strongest effect, but the
actin-capping proteins, cap-1 and -2, the dynein light chain,
dli-1, and the prefoldin subunits (vbp-1, C08F8.1, R151.9,
C17H12.1, F21C3.5, and B0035.4) produced significantly
more progeny than other suppressing clones (Figure 8,
boxed). The capping proteins and prefoldin subunits are
directly or indirectly involved in F-actin assembly, suggest-
ing that the actin cytoskeleton may have an important role in
suppression of dhc-1-induced lethality.
dhc-1(or195ts) Suppressors Have Distinct Effects on
We next determined whether and how knockdown of these
proteins disrupted F-actin organization in wild-type and
dhc-1(or195ts) gonads and embryos. RNAi knockdown of the
dynein light-IC (dli-1), like lis-1(RNAi), suppressed the actin
abnormalities associated with dhc-1(or195ts) in the gonad
and produced some viable progeny. The windows connect-
ing the nuclei to the rachis were regular and small, resem-
bling those observed in wild type (Figure 1), and few nuclei
were present within the rachis (Figure 9). Interestingly, al-
though dli-1 RNAi and lis-1 RNAi similarly improved the
cytoskeleton structure and germline morphology, the level
of embryonic rescue by dli-1 RNAi was much less than
observed for lis-1 RNAi (Figure 8). That lis-1 is more dis-
pensable than dli-1 in the dhc-1(or195ts) background likely
reflects the requirement of dli-1 for dynein function.
RNAi knockdown of the actin-capping proteins cap-1 and
-2 produced indistinguishable results and hereafter will be
referred to as cap(RNAi). cap(RNAi) dramatically increased
the apparent level of F-actin in wild-type and dhc-1(or195ts)
animals (Figures 9 and 10). In both wild-type and dhc-
1(or195ts) animals the F-actin lining the rachis of the gonad
was thickened and ruffled, but in dhc-1(or195ts); cap(RNAi)
animals the windows connecting nuclei with the cytoplasm
were more regular, suggesting that the dhc-1(or195ts) allele
also partially suppresses cap(RNAi) defects. Capping pro-
teins bind and stabilize the barbed plus end of actin, regu-
lating the rate of assembly and disassembly (Hug et al., 1995;
Hopmann and Miller, 2003). Thus, when G-actin levels are
low, cap(RNAi) would be expected to reduce F-actin levels
and when G-actin levels are high, cap(RNAi) would be ex-
pected to increase F-actin levels. The observed increase in
F-actin thus leads us to conclude that there is abundant
G-actin ready for polymerization in the gonad. We also
noticed short actin filaments within the rachis of cap(RNAi)
gonads, filaments which were not present in either wild-
type gonads or in dhc-1(or195ts); cap(RNAi) gonads (Figure
10, arrowheads). The significance of these short actin fila-
ments and their dependence on wild-type dhc-1 is currently
of viable hatched larvae laid by dhc-1(or195ts) animals in two con-
secutive 24-h periods on control bacteria (RNAi vector) or bacteria
producing the indicated dsRNA (see Materials and Methods). The
percent of fertile hermaphrodites is as follows: dhc-1; lis-1 RNAi, n ?
29, 100%; dhc-1; cap-1 RNAi, n ? 14, 85%; dhc-1; cap-2 RNAi, n ? 14,
93%; and dhc-1; dli-1 RNAi, n ? 14, 93%. Note that for single
mutants for each produced no viable progeny. The graph shows
data for select genes, and the table shows numerical data for the
genes selected for further analysis (red rectangle). n ? 3 different
experiments. Error bars, ?SEM.
Suppressors of dhc-1(or195ts) lethality. Average number
Actin Regulators Interact with Dynein
Vol. 21, August 1, 20102715
dhc-1(or195ts) Suppressors Have Distinct Effects on
We showed above that in embryos lis-1 RNAi redistributed
DHC-1(S3200L) protein (Figure 4) and in the germline
caused wild-type DHC-1 to disassociate from perinuculear
regions (Figure 7). To determine whether the mechanisms of
dhc-1(or195ts) suppression were similar for the additional
suppressors, we examined DHC-1(S3200L) localization in
embryos and gonads of RNAi-treated animals. As shown
previously (Yoder and Han, 2001), dli-1 RNAi did not affect
phalloidin-rhodamine, and Hoechst staining of gonads (pachytene region) extruded from dhc-1(or195ts) animals grown at the nonpermissive
temperature (25°C) on bacteria expressing the indicated dsRNA shows that each of the RNAi knockdowns, cap-1, -2, and dli-1, improve
F-actin (red) structure and nuclei (blue) positioning. Microtubule (green) structures are largely unaffected by the treatments. Note the
apparent increase in F-actin content in cap-1 and -2 RNAi-treated animals. Distal (D) and proximal (P) orientations of the gonad is indicated
on the merged image. Scale bar, 10 ?m.
Suppression of dhc-1(or195ts) lethality is linked to the improvement of actin cytoskeleton structure. Anti-?-tubulin antibody,
A. J. Gil-Krzewska et al.
Molecular Biology of the Cell2716
DHC-1 localization in one-cell stage embryos. However, in
the gonad dli-1 RNAi, like lis-1 RNAi, caused DHC-1(wt)
and DHC-1(S3200L) protein to disassociate from the perinu-
clear region and become cytoplasmic (Figure 7). These re-
sults suggest that dli-1 and lis-1 suppress dhc-1(or195ts) by a
similar mechanism. In contrast, cap RNAi caused DHC-1 to
accumulate at F-actin–rich cell junctions in wild-type em-
bryos and to localize very strongly along microtubules (Fig-
ures 5 and 11). Similarly, in the germline, DHC-1 localized
very strongly to the perinuclear region of both meiotic germ
cells and maturing oocytes, where F-actin also accumulated
(Figure 7). However, we were unable to clearly demonstrate
colocalization between F-actin and DHC-1 (Figure 7), and
DHC-1(S3200L) did not accumulate on all F-actin surfaces
(not shown). These observations suggest that DHC-1 local-
ization near F-actin is likely indirect.
Jasplakinolide Increases F-Actin Levels and Relocalizes
DHC-1 in the Gonad
To determine whether the relocalization of DHC-1 associ-
ated with suppression of dhc-1(or195ts) by cap(RNAi) is di-
rectly associated with the cap(RNAi)-induced increase in
F-actin levels, we treated worms and isolated gonads with
jasplakinolide. Jasplakinolide is a peptide isolated from the
marine sponge Jaspis johnstoni that binds to and stabilizes
F-actin in vitro (Bubb et al., 1994). In vivo jasplakinolide
induces F-actin polymerization and aggregation (Senderow-
icz et al., 1995; Holzinger and Meindl, 1997). We found that
the effects of a 30-min jasplakinolide treatment (1 ?M) on the
cytoskeleton of extruded wild-type gonads was similar to
that of lis-1(RNAi) or dhc-1(RNAi) (Figure 12). The F-actin
lining the rachis was ruffled, the windows connecting nuclei
to the rachis were irregular, and many nuclei were within
the rachis (not shown). Strikingly, the jasplakinolide-treated
gonads showed very strong perinuclear DHC-1 localization
(Figure 12). This result suggests that DHC-1 relocalization in
cap(RNAi) animals is caused by changes in the actin cy-
toskeleton. However, we were unable to rescue the dhc-
1(or195ts) defect by jasplakinolde treatment (see Materials
and Methods). This could be due to the inability of dhc-
1(or195ts) to suppress the toxicity of jasplakinolde or may
indicate that cap(RNAi) rescues dhc-1(or195ts) lethality via a
different mechanism. In this regard, capping proteins were
also shown to be a part of the dynactin complex (Schafer et
al., 1994a,b), so the rescue of dhc-1(or195ts) may be unrelated
to the observed relocalization of DHC-1 to F-actin.
thality have distinct effects on cytoskeletal
organization. Anti-?-tubulin antibody, phal-
loidin-rhodamine, and Hoechst staining of go-
nads (pachytene region) extruded from wild-
type (N2) animals grown at 25°C on bacteria
expressing the indicated dsRNA. Each of the
RNAi knockdowns, cap-1, -2, dli-1, and lis-1
affects F-actin (red) organization and the mor-
phology of the rachis, including nuclei (blue)
positioning. cap-1 and -2 RNAi treatments caused
an apparent increase in F-actin content and the
formation of distinct short actin filaments (ar-
rowheads). Microtubule (green) structures are
largely unaffected by the treatments. Distal (D)
and proximal (P) orientations of the gonad is
Suppressors of dhc-1(or195ts) le-
Actin Regulators Interact with Dynein
Vol. 21, August 1, 2010 2717
Here we investigated the genetic and cellular interactions
between dynein and LIS-1 in the C. elegans germline and
early embryo. We found that a temperature-sensitive dynein
heavy-chain mutant dhc-1(or195ts) and loss-of-function of
lis-1 disrupted the F-actin cytoskeleton and cause similar de-
velopmental defects in the germline and early embryo. Unex-
pectedly, the dhc-1(or195ts); lis-1(RNAi) animals produced vi-
able progeny. An RNAi screen of 238 genes predicted to
interact with regulators of the cytoskeleton identified ad-
ditional genes that when depleted suppress dhc-1(or195ts)
lethality. We found that like the dhc-1(or195ts) mutant, de-
pletion of these genes disrupts F-actin organization in the
germline and early embryo. Furthermore, suppression of
dhc-1(or195ts) lethality by depletion of these genes was as-
sociated with restoration of near normal F-actin structures as
well as relocalization of the mutant DHC-1 protein. These
results suggest that F-actin cytoskeleton organization may
play an essential role in the suppression of dhc-1(or195ts)
Genetic studies previously suggested that lis-1 is required
to maintain F-actin structure (Kholmanskikh et al., 2003;
Rehberg et al., 2005). Similarly, we found that LIS-1 as well
as its regulatory target, DHC-1, were required for F-actin
cytoskeletal organization in the pachytene region of the C.
elegans germline. In wild-type gonads F-actin surrounded
each cortically localized nucleus, and the F-actin lining the
rachis was straight and smooth. This highly regular F-actin
organization was disrupted in a similar manner by RNAi
that targeted the expression of genes encoding actin-capping
proteins CAP-1 and -2, by treatment with the F-actin–stabi-
lizing drug jasplakinolide, and by depletion of the microtu-
bule-associated proteins LIS-1, DHC-1, and DLI-1. In each
case the F-actin structure surrounding the rachis was de-
formed, resulting in a ruffled rachis lining, mislocalization of
nuclei into the rachis, and empty and/or multiple nuclei
within single irregularly shaped peripheral actin cages.
These results suggest a role for dynein and the accessory
proteins LIS-1 and DLI-1 in modulating actin dynamics.
We observed that treatments that alter F-actin levels or
organization could suppress the temperature-sensitive dhc-
1(or195ts) allele, suggesting that F-actin dynamics could
modulate dynein activity. Importantly, treatments that sup-
press dhc-1(or195ts) did not simply bypass the F-actin de-
fects, but restore the F-actin cytoskeleton to near wild-type
patterns. This is particularly remarkable, because these treat-
ments in a wild-type background resulted in similar actin-
cytoskeleton defects. The suppression of dhc-1(or195ts) was
also associated with changes in DHC-1 localization in both
the germline and in embryos. However, these changes were
different for lis-1, dli-1, and cap(RNAi). In the germline, de-
pletion of the dynein-associated proteins DLI-1 and LIS-1
caused diffusion of DHC-1 into the cytoplasm, whereas de-
pletion of CAP-1 and -2 caused strong accumulation of
DHC-1 in the perinuclear region. Similar results were ob-
served in one-cell stage C. elegans embryos. In lis-1(RNAi)
and dli-1(RNAi) embryos DHC-1 localization became more
cytoplasmic. In contrast, knockdown of the capping proteins
shifted DHC-1 localization to the periphery of the embryo
where the amount of F-actin also increased. We also ob-
served clear localization of DHC-1 to microtubules in some
embryos. These results demonstrate that changes and/or
restoration of DHC-1 localization may bypass the dhc-
1(or195ts) defect, restoring some dynein function and sup-
animals (dhc-1) at the nonpermissive temperature (25°C) and fed with either control or cap-1 or -2 dsRNA-producing bacteria were stained
with anti-?-tubulin (green), anti-DHC-1 (red), and Hoechst (blue). Individual channels are shown in grayscale, and the merged images are
shown in color. Scale bar, 5 ?m. To better visualize localization of dynein with respect to microtubules, the last column (5?) shows
magnification of the white square–marked area of merged image; scale bar, 1 ?m. cap-1 and -2 knockdown restored dynein localization from
centrosomal location to near wild-type localization along microtubules.
cap-1 and -2 knockdowns restore DHC-1 localization in dhc-1(or195ts) embryos. One-cell stage embryos from dhc-1(or195ts)
A. J. Gil-Krzewska et al.
Molecular Biology of the Cell 2718
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,
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