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 developmental defects in the germline and early embryo. Unexpectedly, the dhc-1(or195ts); lis-1(RNAi) animals produced viable progeny. An RNAi screen of 238 genes predicted to interact with regulators of the cytoskeleton identified additional genes that when depleted suppress dhc-1(or195ts) lethality. We found that like the dhc-1(or195ts) mutant, depletion 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 associated 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) lethality.
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–stabilizing drug jasplakinolide, and by depletion of the microtubule-associated proteins LIS-1, DHC-1, and DLI-1. In each case the F-actin structure surrounding the rachis was deformed, 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 suppress dhc-1(or195ts) did not simply bypass the F-actin defects, but restore the F-actin cytoskeleton to near wild-type patterns. This is particularly remarkable, because these treatments 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, depletion of the dynein-associated proteins DLI-1 and LIS-1 caused diffusion of DHC-1 into the cytoplasm, whereas depletion of CAP-1 and -2 caused strong accumulation of DHC-1 in the perinuclear region. Similar results were observed 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 observed 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 suppressing 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 suspected to regulate actin or microtubule dynamics or function. 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 substantially longer RNAi exposure covering many more developmental 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 knockdowns shift DHC-1(or195ts)
to a more wild-type distribution. Thus, DHC-1(or195ts
) may have normal motor functions 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 suppressed by a dynein heavy-chain mutation that reduces ATPase activity and that causes an increase in dynein localization 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 microtubules. 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 dynein targeting. LIS-1 is involved in targeting dynein to microtubule 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 proteins resulted in strong localization of dynein to areas enriched 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 polymerization–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 suppression of lethality likely requires both the restoration of localization 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 cytoskeleton 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 network. Depletion of these factors in a wild-type background similarly disrupted actin structures, suggesting that despite 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 microtubules; the above depletions caused DHC-1 to redistribute toward a more normal pattern. Therefore, we propose that the common bypass mechanism is decreased dynein processivity 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 cytoskeletal dynamics.