Regulators of the actin cytoskeleton mediate lethality in a Caenorhabditis elegans dhc-1 mutant.
ABSTRACT 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.
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ABSTRACT: Essential genes are critical for the development of all organisms and are associated with many human diseases. These genes have been a difficult category to study prior to the availability of balanced lethal strains. Despite the power of targeted mutagenesis, there are limitations in identifying mutations in essential genes. In this paper, we describe the identification of coding regions for essential genes mutated using forward genetic screens in Caenorhabditis elegans. The lethal mutations described here were isolated and maintained by a wild-type allele on a rescuing duplication.BMC Genomics 05/2014; 15(1):361. · 4.04 Impact Factor
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ABSTRACT: In Drosophila, like in humans, Dystrophin Glycoprotein Complex (DGC) deficiencies cause a life span shortening disease, associated with muscle dysfunction. We performed the first in vivo genetic interaction screen in ageing dystrophic muscles and identified genes that have not been shown before to have a role in the development of muscular dystrophy and interact with dystrophin and/or dystroglycan. Mutations in many of the found interacting genes cause age-dependent morphological and heat-induced physiological defects in muscles, suggesting their importance in the tissue. Majority of them is phylogenetically conserved and implicated in human disorders, mainly tumors and myopathies. Functionally they can be divided into three main categories: proteins involved in communication between muscle and neuron, and interestingly, in mechanical and cellular stress response pathways. Our data show that stress induces muscle degeneration and accelerates age-dependent muscular dystrophy. Dystrophic muscles are already compromised; and as a consequence they are less adaptive and more sensitive to energetic stress and to changes in the ambient temperature. However, only dystroglycan, but not dystrophin deficiency causes extreme myodegeneration induced by energetic stress suggesting that dystroglycan might be a component of the low-energy pathway and act as a transducer of energetic stress in normal and dystrophic muscles.Developmental Biology 04/2011; 352(2):228-42. · 3.64 Impact Factor
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ABSTRACT: Nurses counsel the parents of patients leaving without being seen by a physician (LWBS) about common childhood illnesses. This strategy's impact is not known. To assess the impact of nurse counseling on ED return visits and outcomes for children who LWBS. This retrospective cohort study used the computerized database of a tertiary care pediatric ED. Participants were all triaged children who LWBS between April 1st 2008 and March 31st 2009. Parents who notified nurses of their intention to leave received information and counseling on when to return. This counseling's occurence was this study's exposure of interest. The control group included patients who LWBS without notification and thus were not counseled. The primary outcome was a return visit to the ED within 48h. Triage level and referral status were used as severity indicators. To demonstrate a 2% difference in return visits (α value 0.05, power 80%), 3213 participants were needed per group. During the study period, 60,525 patients consulted the ED and 10,323 LWBS; of these, 4639 (45%) received nurse counseling and 5684 (65%) did not. There was a 2.0% (95% CI 1.0, 3.0) decrease in ED return visit proportions between groups. On multiple logistic regression, the counseled group was less likely to return to the ED within 48h. This study suggests that, of patients who LWBS, those who receive counseling by a nurse have less return visits in the following 48h.International emergency nursing 10/2011; 19(4):173-7.
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 Cell 2708
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, 2010 2709
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 Cell 2710
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, 20102711