Dynamins are targeted to specific cellular membranes that they remodel via membrane fusion or fission. The molecular basis of conferring specificity to dynamins for their target membrane selection is not known. Here, we report a mechanism of nuclear membrane recruitment of Drp6, a dynamin member in Tetrahymena thermophila. Recruitment of Drp6 depends on a domain that binds to cardiolipin (CL)-rich bilayers. Consistent with this, nuclear localization of Drp6 was inhibited either by depleting cellular CL or by substituting a single amino acid residue that abolished Drp6 interactions with CL. Inhibition of CL synthesis, or perturbation in Drp6 recruitment to nuclear membrane, caused defects in the formation of new macronuclei post-conjugation. Taken together, our results elucidate a molecular basis of target membrane selection by a nuclear dynamin and establish the importance of a defined membrane-binding domain and its target lipid in facilitating nuclear expansion.
Topological changes and remodeling of membranes are fundamental processes in cellular physiology. Intricate biological machineries have evolved to facilitate these changes in living cells. Dynamins and dynamin-related proteins (DRPs) comprise a family of large GTPases that catalyze membrane remodeling reactions (Praefcke and McMahon, 2004). Members of the dynamin family are mechano-chemical enzymes that couple the free energy of GTP hydrolysis with membrane deformation, thereby performing important cellular functions ranging from scission of membrane vesicles, cytokinesis, and maintaining mitochondrial dynamics to conferring innate antiviral immunity (Ramachandran and Schmid, 2018). The common features shared by all dynamins and DRPs are the presence of a large GTPase domain (GD), a middle domain, and a GTPase effector domain (GED), which distinguish them from other GTPases (Praefcke and McMahon, 2004). The MD and the GED are involved in oligomerization and regulation of GTPase activity (Ramachandran et al., 2007). The feature that distinguishes DRPs from classical dynamins is the lack of a defined pleckstrin homology domain (PH domain) and a proline-rich domain.
All dynamin proteins undergo rounds of assembly and dis-assembly on the target membrane and tubulate the underlying membrane, which is required for fission or fusion function. The members of this family become associated with lipids on the target membrane and are important for performing cellular functions (Ramachandran and Schmid, 2018). The PH domain in classical dynamins binds to phosphatidyl inositol 4,5 bis-phosphate (PIP2) at the target sites of endocytosis and plays an essential role in vesicle scission during endocytosis (Zheng et al., 1996). All the DRPs lack PH domains and instead possess either lipid binding loops or trans-membrane domains for membrane recruitment or association (Ramachandran and Schmid, 2018). A stretch of positively charged amino acid residues in the lipid binding loops of all known DRPs interacts with the negatively charged head groups of the lipids, and this interaction is important for target membrane association (Rujiviphat et al., 2009; von der Malsburg et al., 2011; Bustillo-Zabalbeitia et al., 2014; Smaczynska-de Rooij et al., 2019; Wang et al., 2019; Yan et al., 2020).
Nuclear remodeling including its expansion is a fundamental process in eukaryotes, the mechanism of which is not well understood in any organism. It requires remodeling of nuclear membrane and incorporation of new materials including lipids and proteins into the existing membrane. Tetrahymena thermophila, a unicellular ciliate, exhibits nuclear dimorphism. Each cell harbors a small, diploid, transcriptionally inactive micronucleus (MIC) and a large, polyploid, transcriptionally active macronucleus (MAC) (Karrer, 2000). During sexual conjugation in Tetrahymena, two cells of complementary mating types first form a pair, followed by a series of complex nuclear events resulting in the loss of the parental MAC. Subsequently, new MIC and MAC are formed through the fusion of haploid nuclei produced from parental micronuclei. The precursors of the new MIC and MAC are identical in size and in genome content at the initial stage of nuclear differentiation. However, two of the four new MICs rapidly enlarge, making the final volume 10- to 15-fold larger, and develop into MACs (Cole et al., 1997). This process calls for a sudden and dramatic expansion of nuclear envelope. Drp6, which is one of the eight DRP paralogs in Tetrahymena, is specifically upregulated when the MICs rapidly expand to form new MACs. Drp6 associates with outer nuclear envelope of both MIC and MAC (Elde et al., 2005), and inhibition of Drp6 function results in a profound deficiency in the formation of new MACs (Rahaman et al., 2008). It has been recently demonstrated that Drp6 functions as an active GTPase and self-assembles into higher order helical spirals and ring structures, and therefore resembles other members of the family (Kar et al., 2018).
In the present study, we have elucidated a mechanism for Drp6 recruitment in the nuclear membrane. Our results reveal that Drp6 directly interacts with membrane lipids and that CL acts as its physiologically important target lipid. Furthermore, we have identified a lipid-binding domain in Drp6 and provide evidence that the domain plays a pivotal role in nuclear association of Drp6 and therefore in nuclear expansion.
All the known dynamin family proteins perform cellular functions by associating with a target membrane. Classical dynamins contain a pleckstrin homology (PH) domain responsible for membrane binding (Figure 1a). Drp6 associates with nuclear envelope and regulates nuclear remodeling in Tetrahymena. However, Drp6 like other DRPs lacks a PH domain or any recognizable membrane-binding domain (Figure 1a). Sequence alignment revealed the presence of a region in Drp6 that is located at the position of the PH domain of classical dynamin (Figure 1b, Figure 1—figure supplement 1a) and that was earlier named the Drp-targeting determinant (DTD) (Elde et al., 2005).
To assess whether the DTD is important for recruitment of Drp6 to target membranes, full-length DRP6 and drp6∆DTD were expressed separately as N-terminal GFP-fusion proteins and their cellular distributions were compared by confocal microscopy. As expected, Drp6 was chiefly present on the nuclear envelope and also on some cytoplasmic puncta (Figure 1c, Figure 1—figure supplement 1b). These cytoplasmic puncta of Drp6 are endoplasmic reticulum derived vesicles (Rahaman et al., 2008). When confocal images of GFP-drp6ΔDTD-expressing cells were analyzed, it was observed that the deletion of DTD resulted in complete loss of nuclear localization, and it was mainly associated with cytoplasmic puncta (Figure 1c, Figure 1—figure supplement 1b). These results clearly demonstrate that DTD is necessary for recruiting Drp6 to the nuclear envelope, but not to cytoplasmic puncta. We next evaluated if the DTD is sufficient for nuclear envelope targeting, by expressing it as a GFP-fusion protein. The confocal images of cells expressing GFP-drp6-DTD showed that GFP-DTD often appeared as cytoplasmic puncta, but that nuclear envelope localization in a subset of cells is detectable albeit less prominently as compared to that of GFP-drp6 (Figure 1c, Figure 1—figure supplement 1b). This suggests that the DTD is able to interact with the nuclear envelope. This interaction appears weaker than for the full-length protein, suggesting that other domains also contribute to nuclear recruitment of Drp6. Similarly, other dynamin family members rely for their targeting on a membrane-binding domain but also depends on other domains such as GD (Vallis et al., 1999; von der Malsburg et al., 2011; Bramkamp, 2012). We conclude that DTD is essential but not sufficient for Drp6 recruitment to the nuclear envelope. In contrast, Drp6 does not require its DTD for targeting to the ER-derived cytoplasmic vesicles.
Recruitment of a protein to a target membrane is achieved either by interaction with membrane lipids or by forming a complex with another membrane protein. The DTD is essential for recruitment of Drp6 to the nuclear envelope and may represent a membrane-binding domain. To test this idea, we generated N-terminal histidine-tagged drp6-DTD and DRP6 for bacterial expression and purification and then used the purified proteins in lipid overlay assays. Drp6 was purified to near homogeneity (Figure 2a) and incubated with total Tetrahymena lipid spotted on nitrocellulose membrane either in the presence or absence of GTP. Drp6 interacts with Tetrahymena lipid with or without GTP (Figure 2b), suggesting that it harbors a lipid-binding domain. To identify the lipids with which Drp6 interacts, we performed the overlay assay using commercially available strips spotted with fifteen different lipids (Echelon Biosciences, USA). The results demonstrated that Drp6 specifically interacts with three phospholipids, namely phosphatidylserine (PS), phosphatidic acid (PA), and cardiolipin (CL) (Figure 2b). In order to find out whether lipid binding is a property of the DTD, we partially purified DTD as an N-terminal his tagged protein (Figure 2a) and used it for the overlay assay. Like the full-length protein, DTD also interacts with all three phospholipids (Figure 2b).
We then looked at lipid binding in the physiologically relevant context of a bilayer, using an in vitro binding assay to liposomes containing 10% PA, 10% PS, or 10% CL. All the liposomes also contained 70% PC and 20% PE. In sucrose density gradients, the recombinant Drp6 co-migrated with all the three types of liposomes, appearing in the top (light) fractions (Figure 2c). Drp6 was not found in the gradient top fractions in the absence of added liposomes (Figure 2c). Similarly, Drp6 also failed to co-migrate with liposomes that contained only PC and PE (Figure 2c). Taken together, our results indicate that Drp6 interacts with membranes in vitro and that this depends on the presence of either PS, PA, or CL. DTD by itself interacts with the same three phospholipids as holo-Drp6, consistent with the idea that DTD is the membrane-binding domain. To further test this idea, we performed sub-cellular fractionation of cells expressing GFP-drp6-DTD or GFP-drp6. As shown in Figure 2d, Drp6 appeared in both soluble and membrane fractions. DTD also appeared in the membrane fraction, suggesting that it can bind to membranes in vivo (Figure 2d). Taken together, these results lead us to conclude that DTD is a membrane-binding domain and requires PS, PA, or CL for association with the membrane.
Dynamin binds to membrane lipids via interaction between its PH domain and the PIP2 head group. A hydrophobic patch in the PH domain of classical dynamin is important for membrane association/insertion (Ramachandran et al., 2009). The sequence similarity between PH domain of human dynamin 1 and corresponding region of Drp6 is very low (Figure 1b). However, the structural similarity is very high among all the dynamin family proteins whose structures are known. Therefore, we generated a three-dimensional model of Drp6 in order to identify the corresponding hydrophobic region in the DTD. The 3-D modeling of Drp6 shows that the structure of DTD is not related to PH domain, but that nonetheless has a hydrophobic patch (aa 553–555) (Figure 3a). To test the importance of this patch, we substituted the first residue, I553, with M. We expressed this mutant allele as an N-terminal GFP-fusion (GFP-Drp6-I553M), and in parallel expressed a GFP-fusion of the wild-type protein (GFP-drp6), and characterized their localization in Tetrahymena by confocal microscopy. While the latter localized mainly on the nuclear envelope with few cytoplasmic puncta, the mutant GFP-drp6-I553M was not visible on the nuclear envelope but was instead exclusively present at cytoplasmic puncta (Figure 3b, Figure 3—figure supplement 1). This difference suggests that the isoleucine at 553rd position is important for nuclear localization of Drp6.
Dynamin and DRPs require binding and hydrolysis of GTP for target membrane localization and membrane remodeling functions. Drp6 hydrolyses GTP in vitro (Kar et al., 2018). To understand whether the mutation at I553 affects GTP binding and/or hydrolysis, we expressed and purified Drp6-I553M and Drp6 as N-terminal histidine-tagged proteins (Figure 4a) and compared their GTPase activities. The GTPase activity of Drp6-I553M (0.061 ± 0.002 nmol/μM/min) was not significantly different from that of Drp6 (0.056 ± 0.003 nmol/μM/min) (Figure 4b). The GTPase activities of both wild-type and mutant proteins were also found to be similar when reactions were carried out for 0–20 min (Figure 4b). We also assessed the Michaelis–Menten constant (Km) and maximum velocity (Vmax) for both these proteins (Figure 4c). The Km of Drp6-I553M (384 µM) was found to be slightly higher than that of Drp6 (180 µM), suggesting a marginal decrease in GTP binding affinity. The mutation did not affect Vmax (0.089 nmol/µM/min for Drp6; 0.0893 nmol/µM/min for Drp6-I553M). Taken together, these results suggest that the defect in nuclear localization of Drp6-I553M is not due to defective GTPase activity.
Another property of DRPs is their ability to assemble and dis-assemble at their target membranes. This involves self-assembly of helical spirals and ring structures and is important for membrane association and membrane remodeling functions. The self-assembly of Drp6 and Drp6-I553M was evaluated by size exclusion chromatography. Drp6 eluted in the void volume as an oligomer containing at least six monomers, as previously observed (Kar et al., 2018; Figure 4d). Similarly, Drp6-I553M also formed higher order structures, as the majority of the protein eluted in the void volume (Figure 4d). A small peak of material eluting near the 150 kDa marker might correspond to a dimer. The breadth of this smaller peak suggests that it consists of a mixture of monomeric and oligomeric structures, with a dimer at the peak fraction. Since the mutant protein was able to form higher order oligomeric structure, we suggest that mutation does not inhibit its self-assembly. However, there might be a difference in the assembly products formed by the wild-type and mutant proteins. To examine this possibility, we compared the ultrastructure of the wild-type and mutant proteins by electron microscopy of negatively stained preparations of the purified recombinant proteins. The Drp6 appeared mostly as large helical spirals, which are similar to structures found in other DRPs (Figure 4e). Ring-like structures were also present, as also found in other members of the family. Similarly, in Drp6-I553M samples, we observed both helical spirals and ring-like structures (Figure 4e). These results suggest that the I553M mutation does not block in vitro oligomerization of Drp6. Taken together, our results suggest that defective nuclear localization of Drp6-I553M is not due to defects in GTPase activity or perturbation of self-assembly.
Dynamin family proteins including Drp6 associate with their target membranes by interacting with specific lipids. To ask whether the I553M mutation might affect these interactions, we used in vitro membrane-binding assays. Recombinant Drp6 and Drp6-I553M proteins were incubated separately with liposomes containing either 10% CL, or PS, or PA. The association of the proteins with liposomes was then judged based on their co-flotation in sucrose density gradients. As can be seen in Figure 5a, Drp6 co-floated with all the three liposomes and appeared on the top fractions, whereas Drp6-I553M co-floated with PS- or PA-containing liposomes but failed to co-float with CL-containing liposomes. Since the mutant retains the ability to associate with PS- and PA-containing liposomes, the overall membrane-binding activity of Drp6 does not depend on I553. Instead, the mutation of I553 specifically inhibits interaction with CL. Based on these results, we infer that I553 in the membrane-binding domain is necessary for CL interactions and that these interactions are important for membrane targeting in vitro.
We then asked whether the interaction between Drp6 and CL was important for nuclear targeting in vivo. Importantly, while the nuclear envelope of animals (Keenna et al., 1970; Kleinig et al., 1971; Sato et al., 1972; Jarasch et al., 1973) lacks CL, it is present in the nuclear membrane of Tetrahymena (Nozawa et al., 1973). To evaluate whether CL is required for nuclear localization of Drp6, we depleted CL from GFP-drp6-expressing cells using pentachlorophenol (PCP), a polychlorinated aromatic compound. PCP is a respiratory uncoupler and a potent inhibitor of CL synthesis (Ono and White, 1971). Within 30 min of PCP treatment, GFP-drp6 dissociated from nuclear envelopes in the majority of cells while remaining associated with cytoplasmic puncta (Figure 5b). Quantitative analysis showed that while GFP-drp6 was localized at the nuclear envelope of all untreated cells, more than 80% of the cells completely lost nuclear localization of GFP-drp6 with the remaining cells showing decreased nuclear localization upon PCP treatment. To check that the nuclear envelope itself remains intact under these conditions, we localized the nuclear pore protein GFP-Nup3/MacNup98B. We found that GFP-Nup3/MacNup98B was clearly associated with nuclear envelopes before or after PCP treatment (Figure 5b). Moreover, PCP treatment does not disrupt membrane structure in general since the distribution of a cortical membrane-binding protein GFP-Nem1D (Shukla et al., 2018) was also not affected by this treatment (Figure 5b). These results therefore suggest that the delocalization of GFP-drp6 upon PCP treatment is due to loss of CL.
If the defect in localization of Drp6-I553M is due to a defect in CL-dependent targeting, one might expect that the defect would be suppressed in the presence of the wild-type protein, since the mutant protein would co-assemble with the correctly targeted wild type. To test this idea, we co-expressed GFP-drp6-I553M and mCherry-drp6. mCherry-drp6 colocalized almost entirely with GFP-drp6-I553M and was targeted to nuclear envelopes as well as cytoplasmic puncta, strongly suggesting that the mutant protein is able to co-assemble with the wild-type protein (Figure 5c). This result also reinforces the earlier conclusion that the mutation does not affect the overall structure of the protein.
After establishing the necessity of the I553 residue and CL in Drp6 nuclear recruitment, we addressed if the loss of nuclear recruitment of Drp6-I553M is specifically due to the presence of methionine at this position. To test this, we replaced I553 with alanine in the GFP-drp6-I553A protein and expressed it in the cell. Confocal microscopic analysis demonstrated that, similar to GFP-drp6-I553M, GFP-drp6-I553A also lost its association with nuclear envelope and was detected mostly as cytoplasmic puncta (Figure 6a, Figure 6—figure supplement 1). As expected, the wild-type GFP-drp6 was chiefly localized in the nuclear envelope under the same experimental conditions. This result reiterates our earlier conclusion that the I553 residue is critical for nuclear recruitment of Drp6. We further confirmed that the loss of Drp6-I553A recruitment to the nuclear envelope is specifically due to loss of its interaction with CL by performing floatation assay (Figure 6b). For this purpose, we purified recombinant Drp6-I553A protein and tested its binding to liposomes containing either CL, PS, or PA. Western blot analysis showed that Drp6-I553A lost interaction with CL liposomes while retaining its interaction with PS and PA liposomes (Figure 6b). To rule out that the loss of nuclear association is not due to the defect in GTPase activity or self-assembly, we performed size exclusion chromatography and GTP hydrolysis assay. When the purified recombinant Drp6-I553A protein was subjected to size exclusion chromatography, it eluted primarily in the void volume as higher order oligomeric structures (Figure 6c). A small fraction appearing as a mixture of monomers and oligomers with a peak around the dimer was also observed in the chromatogram. The GTPase assay results suggested that the I→A substitution does not affect its activity as assessed by the rate of GTP hydrolysis (Figure 6d,e). Based on these results, we conclude that the loss of Drp6-I553A nuclear recruitment is not due to the defect in GTPase activity or in the self-assembly of Drp6. The results also establish that Ile at the 553rd position is essential for Drp6-CL interactions and is a critical determinant of Drp6 nuclear recruitment.
We next examined whether the loss of interaction between the mutant proteins and CL is due to a change in the overall folding or conformation of Drp6. To assess overall folding, circular dichroism (CD) spectra of the mutants were compared with that of wild-type protein (Figure 6f). The analysis of far-UV CD spectra showed that Drp6 contains both α-helix and β-sheets as secondary structures with an ellipticity minima at 222 nm. Similar spectra were observed for I553M and I553A mutants. These results suggest that the mutant proteins were able to fold correctly. To rule out any change in the three-dimensional conformation due to the amino acid substitutions, we performed fluorescence spectroscopic analysis using intrinsic fluorescence of the lone tryptophan present in the Drp6 protein. The fluorescence spectra of the wild-type Drp6 showed a peak at around 332 nm, suggesting that the tryptophan residue is mostly buried (Figure 6g). The fluorescence spectra of I553M and I553A mutants did not show significant difference from that of wild type, suggesting that the overall structure of the proteins did not change due to mutations. To further assess the conformation around the I553 residue, acrylamide quenching experiments were performed using intrinsic tryptophan fluorescence. Since the single tryptophan (W548) is located only five amino acid residues apart from the I553, it allowed us to monitor the effect of mutations on the local conformation around this isoleucine residue. The accessibility of the tryptophan was measured by determining the quenching constant (kq) for wild-type protein as well as the mutant proteins (Figure 6h). The results showed comparable values of kq (1.01 M−1 ns−1 for Drp6; 1.11 M−1 ns−1 for Drp6-I553M and 1.04 M−1 ns−1 for Drp6-I553A) for all the three proteins, suggesting that there is no major change in the local conformation due to mutations. These results lead us to conclude that the loss of CL interaction with I553A or I553M mutant is not due to a change in overall folding or local conformation of the mutant proteins. Taken together, it can be concluded that a single isoleucine residue in the membrane-binding domain is essential for interaction with CL and targeting Drp6 to the nuclear membrane.
We next investigated the role of two amino acid residues (E552 and M554) at the vicinity of I553 in CL binding and nuclear association. For this purpose, two independent mutants drp6-E552D and drp6-M554L were expressed either as N-terminal GFP-fusion proteins to assess their localizations in Tetrahymena cells or as N-terminal histidine fusion proteins for determining CL binding in vitro. Confocal microscopic analysis of the Tetrahymena cells expressing either GFP-drp6-E552D or GFP-drp6-M554L showed that, in addition to cytoplasmic puncta, both the mutant proteins also associated with nuclear envelope (Figure 7a). Floatation assay was performed to evaluate whether these two mutants also bind to CL-containing membrane in vitro. Both Drp6-E552D and Drp6-M554L appeared on the top fractions in the floatation experiments, suggesting their binding to CL (Figure 7b). Therefore, we conclude that the amino acid residues neighboring I553 do not determine the specificity for CL binding and nuclear recruitment of Drp6. Thus, the isoleucine at the 553rd position confers specificity for CL mediated nuclear recruitment of Drp6.
Previously, it has been shown that Drp6 is essential for MAC development in Tetrahymena (Rahaman et al., 2008). We have now established that interaction of Drp6 with CL is important for nuclear recruitment. Therefore, we hypothesized that inhibition of CL-Drp6 interaction would inhibit Drp6 function in MAC development. We took two independent approaches to perturb the interaction between CL and Drp6 and assessed the effect on MAC development. In the first approach, CL was depleted by treating cells at a stage prior to MAC development with PCP and then measuring the efficiency of new MAC formation in conjugating cells (Figure 8a). Quantitative analysis showed that while 71 ± 3.6% of the conjugants developed MACs in the control pairs, only 24 ± 1.7% developed MACs in the PCP-treated pairs (Figure 8a). In the second approach, we perturbed CL–Drp6 interaction by treating conjugants with nonyl acridine orange (NAO). NAO interacts with CL with very high affinity and has been used in mammalian cells to block interactions between CL and mitochondrial proteins involved in electron transport (Maftah et al., 1990). Exposing conjugants to NAO significantly inhibited new MAC development (Figure 8a).
In conjugating Tetrahymena, MAC development is not the only phenomenon requiring nuclear expansion. At a prior stage, the germline micronuclei (MICs) show dramatic elongation (Cole et al., 1997). We found that PCP treatment did not affect the frequency of elongation, that is, the percentage of pairs showing elongated MICs, but did produce a decrease in the extent of MIC elongation (Figure 8a). This inhibition of elongation had no detectable consequences for the subsequent stage of MIC meiosis. Treatment with NAO had no measurable effect on MIC elongation (Figure 8a) or the subsequent meiosis. These results are consistent with the idea that the key requirement for CL is during MAC expansion.
We next reasoned that if interaction of Drp6 with CL is important for MAC expansion, then over-expression of the isolated Drp6 membrane-binding domain might competitively inhibit the interaction and block Drp6 function during MAC expansion. To test this possibility, we expressed GFP-drp6-DTD in Tetrahymena and then allowed the cells to conjugate with a Drp6 wild-type strain. We measured MAC development in these pairs, and in pairs from a parallel WT x WT cross, at 8 hr during conjugation. In the control cross, more than 75% of pairs developed new MACs. In striking contrast, only 4–5% of pairs developed normal MACs in the pairs that included GFP-drp6-DTD-expressing cells (Figure 8b). Therefore, MAC development was almost completely blocked when GFP-drp6-DTD-expressing cells comprised one of the conjugation partners (Figure 8b).
In similar experiments, we also asked whether the expression of GFP-drp6∆DTD might have a dominant-negative inhibitory effect on Drp6 function. Indeed, we found that in pairs where one cell over-expressed the ∆DTD construct, the pairs showed inhibition of MAC development that was similar to that induced by expression of the isolated DTD domain (Figure 8b). Neither the expression of GFP-drp6-DTD nor GFP-drp6∆DTD significantly reduced the fraction of pairs showing elongated MICs (Figure 8b).
In conclusion, prior experiments with DRP6 gene knockout pointed to a specific function in MAC development. Our current results from over-expression of mutant alleles are consistent with this idea. Taken together, our results support a model in which interaction of Drp6 with CL, for which a single isoleucine residue acts as a key determinant, is critical for nuclear targeting and therefore for MAC expansion.
Drp6 is a nuclear dynamin and is involved in nuclear remodeling (Rahaman et al., 2008). In the present study, we have identified a membrane-binding domain in Drp6 that directly interacts with lipids. Inhibition of CL synthesis blocks nuclear localization of Drp6, suggesting that it plays a critical role in Drp6 recruitment. Further evidence of CL interaction determining nuclear localization of Drp6 comes from the mutation of isoleucine at the 553rd position. The mutant Drp6 proteins lose their nuclear localizations with concomitant loss of interaction with CL without affecting other properties such as GTPase activity and self-assembly into rings/helical spirals. The GFP-drp6-I553M recruited to the nuclear envelope only when co-expressed with wild-type mCherry-drp6, indicating that the mutant protein co-assembles with the wild type (Figure 5c). These results suggest that Drp6 molecules self-assemble on the nuclear envelope, and localizing the oligomer to the envelope does not require all subunits interact with CL. We further demonstrate that Ile at position 553 in the DTD is the sole residue critical for CL interactions, whereas the adjacent two amino acid residues (Glu at 552nd position and Met at 554th position) are not essential for CL interaction specificity. Taken together, these results suggest that a single isoleucine residue in the membrane-binding domain specifies the recruitment of Drp6 to the nuclear membrane.
We investigated the significance of CL in the nuclear remodeling function of Drp6. Inhibition of CL synthesis as well as perturbation of its interaction with Drp6 phenocopy the loss-of-function phenotype of Drp6, suggesting a role for CL in Drp6-mediated nuclear remodeling. Over-expression of drp6-DTD or drp6ΔDTD inhibits MAC expansion. Since DTD interacts with lipid including CL, the inhibition of MAC expansion is expected to be by competing with Drp6-CL interaction, concomitantly inhibiting Drp6 recruitment to the nuclear envelope. Inhibition of nuclear expansion by ΔDTD can be due to the inhibition of Drp6 localization on the nuclear envelope by forming a heterogenic complex as it lacks membrane-binding domain and does not associate with nuclear envelope. Based on these results, it can be concluded that CL acts as a molecular determinant in recruiting Drp6 on the nuclear envelope to perform nuclear remodeling function.
Drp6 is involved in nuclear expansion, which requires the incorporation of new lipids into the existing nuclear membrane, suggesting a membrane fusion function for Drp6. Consistent with this, we recently observed that Drp6 is able to perform membrane fusion in vitro (our unpublished results). Membrane fission or fusion involves exchange of lipids between two juxtaposed bilayers, and optimum membrane fluidity is likely to be essential for the exchange of lipids between adjacent leaflets. CL is known to facilitate the formation of apposed bilayers as well as to enhance membrane fluidity (Unsay et al., 2013). Dynamin proteins remodel membrane and bring bilayers to the vicinity during fission or fusion of membranes (Praefcke and McMahon, 2004). Therefore, interaction of Drp6 with CL may enhance bilayer interaction and membrane fluidity. Taking together, it is reasonable to conclude that Drp6-CL interaction on the nuclear envelope facilitates nuclear expansion by enhancing membrane fusion and hence is essential for macronuclear expansion.
Drp6 interacts with three different lipids namely CL, PA, and PS (present study). These interactions with multiple lipids might explain the localization of Drp6 in multiple sites (Figure 9). The target specificity is often determined by the interaction of the membrane-binding domain with the specific lipids on the membrane. Although DRPs including Drp6 lack PH domain, they harbor a membrane-binding domain at the corresponding location (Figures 1a and 3a; Ramachandran and Schmid, 2018). The sequence diversity in this domain might explain the diverse functions of the family members on different target membranes. While PIP2 present in the plasma membrane associates with endocytic dynamin at the neck of vesicles, CL exclusively present in the mitochondria is recognized by the dynamins possessing mitochondrial remodeling function (Francy et al., 2017; Kameoka et al., 2018). It is important to note that the nuclear envelope of Tetrahymena contains 3% CL (Nozawa et al., 1973), and Drp6 (which is specifically present in the ciliate Tetrahymena) has evolved to interact with CL for specific recruitment to the nuclear envelope. In addition to nuclear envelope, Drp6 is also associated with ER vesicles (Rahaman et al., 2008). Localization of Drp6-I553M/Drp6-I553A mutants on ER vesicles and their ability to interact with PS and PA on the membrane suggest that Drp6 localization on ER is dependent on either PA or PS or combination of both. Considering the abundance of PA on the ER membrane (Pillai et al., 2017a; Pillai et al., 2017b; Zegarlińska et al., 2018), it could be argued that PA is involved in recruitment of Drp6 to ER. We also observed localization of Drp6 on plasma membrane (Figure 9), and since PS is also present in the plasma membrane (Kay et al., 2012), it is possible that Drp6 associates with plasma membrane via its interaction with PS. Although further experiments are required to find out the role of PS and PA in the recruitment of Drp6 in plasma membrane and ER, our results clearly show that lipid molecules play critical role in compartmentalizing the localization of Drp6 where CL shifts the dynamics from ER vesicles to nuclear envelope.
As mentioned earlier, I553 in the Drp6 membrane domain is critical for conferring specificity to Drp6 for recognizing CL on the nuclear envelope. The importance of I553 suggests the importance of hydrophobic patches for target membrane specificity, since CL is known to interact strongly with hydrophobic residues (Planas-Iglesias et al., 2015). This is substantiated in the endocytic dynamin that uses a hydrophobic region including isoleucine at the 533rd position in the PH domain for insertion into the target membrane (Ramachandran et al., 2009). Our results on isoleucine mutation within the membrane domain also suggest that the presence of a similar hydrophobic patch in Drp6 that is important for membrane interaction specifically via CL present on the nuclear envelope. However, hydrophobicity is not the sole determinant for the specific interaction with CL since methionine, which is also a strong hydrophobic residue, does not interact with CL when substituted for isoleucine (Figure 5a). Therefore, it is conceivable that, in addition to hydrophobicity, the side chain of isoleucine plays a critical role in conferring the specificity for the recruitment to the target membrane via interaction with CL. Although residues important for target membrane selection have been identified in many dynamin proteins, they involve a stretch of positively charged amino acids for recognizing anionic head groups of several lipids including CL, PS, and PIP2 (Salim et al., 1996; Achiriloaie et al., 1999; Vallis et al., 1999; Rujiviphat et al., 2009; von der Malsburg et al., 2011; Bustillo-Zabalbeitia et al., 2014; Smaczynska-de Rooij et al., 2016; Wang et al., 2019). However, it is not known how different dynamin proteins distinguish different lipids solely based on ionic interaction. In the present study, we demonstrate that a single isoleucine in the membrane-binding domain of Drp6 provides the specificity for CL. This isoleucine residue, however, does not influence Drp6 interaction with PS or PA, hence distinguishes among different negatively charged lipids. This is the first example of any dynamin protein in which a single amino acid site is shown to be critical for specific lipid interactions, thereby providing an additional target membrane (nuclear membrane) binding property to the protein. In conclusion, our results provide the underlying mechanism of target membrane selection by a nuclear dynamin and underscore the importance of CL interaction with a single amino acid residue in the Drp6 membrane-binding domain in facilitating nuclear expansion.
Tetrahymena thermophila CU428 and B2086 strains were obtained from Tetrahymena Stock Center (Cornell University, USA). Cells were cultured in SPP medium (2% proteose peptone [BD, USA], 0.2% glucose, 0.1% yeast extract, and 0.003% ferric EDTA) at 30°C under shaking at 90 rpm. For conjugation, mating type cells were grown in SPP media to a density of 3 × 105 cells/ml, washed and resuspended in DMC media (0.17 mM sodium citrate, 0.1 mM NaH2PO4, 0.1 mM Na2HPO4, 0.65 mM CaCl2, 0.1 mM MgCl2), and incubated at 30°C, 90 rpm for 16–18 hr. To initiate conjugation, starved cells of two different mating types were mixed and incubated at 30°C without shaking. All the reagents were purchased from Sigma–Aldrich unless mentioned otherwise.
The Drp6∆DTD lacking aa 517–600 was created by overlap PCR using a set of four oligonucleotides in a two-step PCR method. Drp6-E552D, Drp6-I553M, Drp6-I553A, and Drp6-M554L were generated by site-directed mutagenesis using Quick Change protocol (Stratagene). For expression in Tetrahymena, rDNA-based vector pVGF or pIGF was used. While the PCR products of Drp6 (aa 1–710) and Drp6-DTD (aa 517–600) were inserted between XhoI and ApaI restriction sites of pVGF, the Drp6-E552D, Drp6-I553M, Drp6-I553A, Drp6-M554L, and Drp6∆DTD were introduced into pIGF using Gateway cloning strategy (Invitrogen) using the manufacturer’s protocol and were expressed as N-Ter GFP-tagged fusion proteins. All the constructs were confirmed by sequencing. Conjugating wild-type Tetrahymena cells were transformed with these constructs by electroporation, and the transformants were selected using 100 µg/ml paromomycin sulfate. For the co-expression studies, mCherry-drp6 was generated by introducing mCherry sequences between PmeI and XhoI sites followed by Drp6 sequences between XhoI and ApaI sites of NCVB vector. Co-transformants were generated by biolistic transformation of linearized mCherry-drp6 nucleotide sequences into the cells expressing GFP-drp6-I553M and were selected in the presence of 60 µg/ml blasticidine and 120 µg/ml paromomycin sulfate supplemented with 1 µg/ml cadmium chloride.
Cells were grown to a log phase (2.5–3.5 × 105 cells/ml), and expression was induced by adding cadmium chloride at concentration of 1 µg/ml for 4 hr. Cells were harvested at 1100 g, fixed with 4% paraformaldehyde for 20 min at RT, washed and resuspended in 10 mM HEPES, pH 7.5, and DAPI stained before imaging.
For expression in Escherichia coli, the amplified PCR products of Drp6 and Drp6-DTD were cloned into pRSETB using BamHI and EcoRI sites. The Drp6-E552D, Drp6-I553M, Drp6-I553A, and Drp6-M554L were cloned in pRSETB vector using Quick Change protocol (Stratagene). The resulting constructs were transformed into chemically competent E. coli C41(DE3) cells and transformants were inoculated into LB broth supplemented with 100 μg/ml ampicillin and grown at 37°C till the OD600 reached 0.4. The cultures were then shifted to 18°C and expression was induced after 1 hr by adding 0.5 mM IPTG (Sigma) and kept for 16 hours at the same temperature before harvesting the cells. The harvested cells were resuspended inice-cold buffer A (25 mM HEPES pH 7.5, 300 mM NaCl, 2 mM MgCl2, 2 mM β-mercaptoethanol, and 10% glycerol) supplemented with EDTA-free protease inhibitor cocktail (Roche) and 100 mM phenyl methyl sulfonyl fluoride, lysed by sonication, and the lysates were centrifuged at 52,000 g for 45 min at 4°C. The supernatants were incubated with Ni-NTA agarose resin (Qiagen, Germany) for 2 hr before washing with 100 bed volume buffer A supplemented with 50 mM imidazole. The bound proteins were eluted with 250 mM imidazole in buffer A. The purified proteins were checked by Coomassie-stained SDS–PAGE gels, and the purity was assessed by Image J analysis (NIH, USA). The fractions containing the purified proteins were pooled, dialyzed with buffer A, and concentrated using Amicon ultra-15 filters (Merck-Millipore, Germany). Protein concentration was estimated by Bradford assay (Bio-Rad Laboratories, USA).
Samples were subjected to SDS–PAGE gel, and the proteins were transferred to PVDF membrane. Membrane was blocked with 2% bovine serum albumin (BSA) in TBST (50 mM Tris–Cl, 150 mM NaCl, pH 8.0%, and 0.05% Tween 20) for 1 hr. The blot was then incubated with HRP-conjugated anti-His monoclonal antibody (1:5000) and detected with supersignalfemto substrate (Thermoscientific, USA) using ChemiDoc imaging system (Bio-Rad Laboratories, USA).
Tetrahymena cells expressing GFP-drp6 or GFP-DTD were lysed in 500 µl of ice-cold lysis buffer containing 25 mM Tris–Cl pH 7.5, 300 mM NaCl, 10% glycerol supplemented with E-64, pepstatin, aprotinin, PMSF, and protease inhibitor cocktail (Roche) by passing through a ball-bearing homogenizer with a nominal clearance of 0.0007 in. The resulting lysates were centrifuged at 16,000 g for 15 min at 4°C, the supernatant was collected as soluble protein fraction, and the pellet containing the membrane fraction was resuspended in 500 µl lysis buffer. The proteins in both soluble fraction and membrane fraction were separated in 12% SDS–PAGE gel and analyzed by western blotting using anti-GFP polyclonal antibody (1:4000; Sigma–Aldrich).
Total Tetrahymena lipid was extracted from growing Tetrahymena cells (5 × 105 cells/ml) following the method by Bligh and Dyer, 1959. Drops of 5 µl in chloroform were spotted on the nitrocellulose membrane and incubated with His-drp6 (90 µg/ml) in GTPase assay buffer in the presence or absence of 1 mM GTP for 1 hr. In control experiments, BSA was used in place of His-drp6. The assay using membrane lipid strips (P-6002, Echelon Biosciences, USA) spotted with 100 pmol of 15 different lipids were used according to the manufacturer’s instruction. The binding of proteins was detected by western blot analysis using anti-His monoclonal antibody.
Lipids (Avanti Polar) were dissolved in analytical grade chloroform, and liposomes were prepared using 2.5 mg total lipid in 1 ml chloroform. The liposomes contained 70% PC and 20% PE along with either 10% CL or 10% PA or 10% PS. A thin dry film was obtained by drying the solution in a round bottom flask, and solvent was completely removed in a lyophilizer. Liposomes were made by rehydrating the film in buffer A (25 mM HEPES pH 7.5, 2 mM MgCl2, 150 mM NaCl) pre-warmed at 37°C. The resuspended solution was extruded 17–21 times through extruder (Avanti Polar) using filter with 100 nm pore, and the size distribution was measured by DLS in Malvern Zetasizer Nano. For floatation assay, 1 µM protein was incubated with 0.5 mg liposomes in buffer A supplemented with 1 mM GTP for 1 hr at RT. Sucrose was added to the reaction mixture (final sucrose concentration 40%), placed at the bottom of a 13 ml ultra-centrifugation tube, and overlaid with 2 ml each of 35%, 30%, 25%, 20%, 15%, and 0% sucrose solutions in the same buffer. The gradient was subjected to ultra-centrifugation in Beckman Coulter ultra-centrifuge at 35,000 RPM for 15 hr at 4°C using SW41 rotor. Fractions (1 ml each) were collected from top and detected by western blotting using anti-His HRP-conjugated monoclonal antibody (1:5000).
The GTP hydrolysis activity of recombinant Drp6, Drp6 I553M, and Drp6-I553A was measured in a colorimetric assay using Malachite Green-based phosphate assay reagent (BIOMOL Green, Enzo Life Sciences, USA). The GTPase assay (20 µl in 25 mM HEPES pH7.5, 15 mM KCl, 2 mM MgCl2) was performed in the presence of 1 mM GTP (Sigma) using 1 μM protein for 0–30 min at 37°C. The reaction was stopped by adding 5 μl of 0.5 M EDTA, and absorbance was measured at 620 nm. For measuring KM and Kcat, reactions were performed for 10 min at 37°C in triplicate using varying concentrations of GTP (50 μM–2000 μM). The values obtained from three independent experiments were plotted and analyzed using GraphPad Prism7 software. The statistical analysis was performed by using unpaired t-test.
Size exclusion chromatography was performed on the Superdex 200 GL 10/300 column (GE Life Sciences, USA) using Akta Explorer FPLC system (GE Healthcare, USA), which was calibrated with standard molecular weight markers (Sigma–Aldrich, USA). Five hundred microliters of protein (0.5 mg/ml) in buffer A was loaded onto the pre-equilibrated column and was run at 0.5 ml/min. The chromatogram was recorded by taking absorbance at 280 nm.
Purified recombinant Drp6 or Drp6 I553M (1 μM) was incubated with 0.5 mM GTPγS in 25 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM MgCl2 for 20 min at room temperature and was adsorbed for 2 min onto a 200 mesh carbon coated Copper grid (Ted Pella, Inc, USA). The grid was stained with a drop of 2% freshly prepared uranyl acetate (MP Biomedicals, USA) for 2 min and dried at room temperature for 10 min. The electron micrographs were collected on a FEI Tecnai G2 120 kV electron microscope.
Tetrahymena cells were fixed with 4% paraformaldehyde (PFA) in 50 mM HEPES pH 7.5 for 20 min at RT and were collected in 10 mM HEPES pH 7.5 after centrifugation at 1100 × g. The fixed cells were stained with DAPI (0.25 ug/ml) (Invitrogen, USA) and washed with 10 mM HEPES pH 7.5 before imaging. The images were collected in a Zeiss LSM780 or Leica DMI8 confocal microscope.
CD measurements were recorded at 145 µg/ml protein in 25 mM HEPES pH 7.5, 300 mM NaCl, 2 mM MgCl2, 2 mM β-mercaptoethanol using a Jasco J-1500 instrument (Jasco Inc, USA). Ellipticity (millidegrees) was measured between 260 and 205 nm. Mean residue ellipticity ([θ]MRW in deg cm2 dmol−1) for each wavelength was calculated by using the following formula:
where θ is the ellipticity in mdeg, Mr is the molecular weight of the protein in Da, c is protein concentration in mg/ml, l is the path length in centimeter, and NA is the number of amino acid residues of the protein.
For the fluorescence spectroscopy experiments, 300 µl of 0.2 µM protein in 25 mM HEPES pH 7.5, 300 mM NaCl, 2 mM MgCl2, and 2 mM β-mercaptoethanol were measured by excitation at 295 nm (2 nm band pass) in a 10 mm quartz cuvette using FLS-1000 (Edinburgh Instruments Ltd., UK). For the fluorescence quenching experiments, different acrylamide concentrations (0–400 mM) were added to 300 µl of 1.5 µM protein sample, and the spectra were recorded in the Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA). The emission spectra were collected from 310 to 410 nm (10 nm band pass), and the values at 332 nm (the emission peak) were analyzed using Stern–Volmer equation, expressed as follows:
where F0 is the fluorescence in the absence of quencher and F is the fluorescence in the presence of quencher. Q denotes the concentration of the quencher in M. Ksv is the Stern–Volmer constant. The obtained values were plotted using GraphPad Prism 7, and Ksv was calculated from the slope of the graph. The quenching constant was calculated using the equation:
Where kq is the quenching constant (in Mol−1 ns−1) and τ is the life time (in ns) of tryptophan. The value of τ is considered to be 2.7 ns as reported earlier (Ronda et al., 2018).
The growing Tetrahymena cells either expressing GFP-drp6 or GFP-Nup3/MacNup98B (a kind gift from Prof. Douglas Chalker, Washington University in St. Louis; Malone et al., 2008; Iwamoto et al., 2009) or GFP-Nem1D were treated with 10 µM PCP in DMSO for 30 min before fixing with 4% paraformaldehyde. The conjugation pairs were treated with either 30 µM PCP or 0.5 µM NAO (Invitrogen, USA) after 2.5 hr, 4.5 hr, or 7.5 hr post-mixing and were fixed after 30 min of addition. The cells were stained with DAPI (0.25 µg/ml) before imaging. The statistical analysis was performed by using unpaired t-test.
All data generated or analysed during this study are included in the manuscript and supporting files.
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Vivek MalhotraSenior and Reviewing Editor; The Barcelona Institute of Science and Technology, Spain
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
[Editors' note: this paper was reviewed by Review Commons.]
Decision letter after peer review:
Thank you for submitting your article "Cardiolipin targets a dynamin related protein to the nuclear membrane" for consideration by eLife. Your article has been reviewed by two external experts and the evaluation has been overseen by a Vivek Malhotra as the Senior Editor.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
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The paper reports the involvement of isoleucine 553 in targeting Drp6 to cardiolipin containing nuclear membrane. The data are interesting, but there is no mechanistic understanding of how a single amino acid can target this protein so specifically to cardiolipin enriched membranes
The authors are strongly requested to address the issues that were raised in the previous review. The authors state in their rebuttal that they plan to address them in a timely manner.
The additional request of one reviewer that should be addressed is to test the involvement of residue 552 and 554 to highlight the significance of isoleucine in position 553 in targeting Drp6 to cardiolipin.https://doi.org/10.7554/eLife.64416.sa1
We thank all the reviewers for their critical evaluation and excellent suggestions to improve the manuscript. We have made all the changes suggested by the reviewers and performed the experiments to address the various concerns raised. Please find below our response to the reviewers’ comments:
1) This is an interesting study from the Rahaman group that identifies cardiolipin (CL) as a potential binding target for Drp6 recruitment to the nuclear membrane in Tetrahymena (that has a unique nuclear remodeling program). In addition, they identify a residue, I553 in the DTD region, which they claim is a key residue involved in specific CL interactions. While the experiments themselves are technically sound, and are well performed and controlled, I don't find the major conclusion that I553 is involved in direct CL interactions justified or well rationalized. By their own admission (in the Discussion), the conservative mutation I553M may perturb local folding and may indirectly affect CL interactions. There is no test of DTD folding with and without the I553M mutation, nor are there other mutations (e.g. I553A and in the vicinity) tested. CD experiments in the absence and presence of CL-containing membranes will likely yield information on the impact of the I553 mutations, while DLS experiments would inform on the hydrodynamic properties (overall 3D fold) of the DTD and the impact of these mutations. CL interactions generally involve a combination of electrostatic and hydrophobic forces. Where do the electrostatic interactions come from? Why would an Isoleucine to Methionine mutation affect the hydrophobic component, even if I553 is the key hydrophobic residue?
We thank the reviewer for appreciating our work. We agree that the exact mechanism of how I553 provides specificity to cardiolipin binding was not addressed adequately in the previous manuscript. We have now performed several experiments to address this issue. Our results clearly show that isoleucine at 553 position is critical for cardiolipin binding and nuclear recruitment of Drp6 and the interaction is direct.
To show that the interaction is direct we have generated another mutant I553A and performed all the experiments including nuclear localization, cardiolipin binding, self-assembly, GTPase activity. Similar to I553M, I553A mutant also failed to bind cardiolipin and to associate with nuclear membrane without affecting GTPase activity and self-assembly of Drp6.These results confirm that isoleucine is critical at this position. We have included these results in Figure 6A-E and Figure 6—figure supplement 1 in the revised manuscript.
To demonstrate that overall folding is not changed due to mutations at this position, we have performed CD spectroscopic analysis and found that mutations didn’t affect overall folding. We have also included these results in Figure 6F in the revised manuscript.
Additionally, we have performed fluorescence spectroscopy using the only tryptophan (W548) residue present nearby I553. The tryptophan fluorescence emission spectra of mutants (I553M and I553A) were similar to that of wildtype Drp6 suggesting there is no major change in conformation due to mutations. To evaluate any minor change in the local conformation due to mutations, we have performed acrylamide quenching experiments using the intrinsic tryptophan fluorescence. The results show that quenching constants of both the mutants were similar to that of wildtype Drp6, and therefore we conclude that the local conformation is not changed due to mutations. These results are included in Figure 6G and 6H in the revised manuscript.
Electrostatic interactions are important for all the phospholipids while interacting with protein and is expected to come from other amino acid residues which are positively charged. Electrostatic interaction may contribute to the affinity of the interaction by providing additional binding energy. But considering its universal nature of interaction with all the phospholipids, it cannot give specificity for a specific lipid and hence would not discriminate among different phospholipids.
Regarding the hydrophobic component, the reviewer is correct that both are strong hydrophobic amino acids and loss of I553M interaction with cardiolipin may not be due to change in hydrophobicity. This is now explicitly mentioned in the manuscript. Therefore, it appears that the side chain of isoleucine at 553 position is important for binding to cardiolipin.
Reviewer #1 (Significance (Required)):
2) The addressed phenomenon is restricted to Tetrahymena and may not have far reaching implications. Regardless, the identification of CL as a binding target for Drp6 at the nuclear membrane of this organism is in itself significant. The conclusion that I553 is the key CL binding residue is however not warranted. Additional experiments are needed to dissect how this residue impacts CL interactions and examine whether the observed effect is direct or indirect.
We thank the reviewer for appreciating the significance of this work. We agree that our data is Tetrahymena specific. However, we believe that the study is potentially relevant for all the proteins whose association with target membranes depend on cardiolipin including many cardiolipin interacting DRPs (such as DRPs involved in biogenesis and maintenance of mitochondria).
As mentioned in the previous section, all the recent results now reinforce that the isoleucine is critical for binding cardiolipin and the interaction is direct.
3) The writing is not clear in some parts and may require a round of language editing. There are no issues with reproducibility.
We have rewritten the manuscript to the best of our abilities and corrected the language.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Dynamin is a GTPase superfamily protein involved in membrane fusion and division. This paper focused on Drp6, one of the eight dynamin superfamily proteins of Tetrahymena, and analyzed its nuclear envelope localization mechanism by a combination of in vivo cytogenetical analysis and in vitro biochemical analysis for the various mutant Drp6 proteins. Results showed that a specific amino acid residue (isoleucine at the 553rd) in the membrane binding domain of Drp6 was required for its nuclear membrane localization, but this residue is not required for ER/endosome localization and GTPase activity. Furthermore, in vitro floating analysis using centrifugation indicated that Drp6 specifically bound to the cardiolipin at the 553rd isoleucine residue and this binding was required for Drp6's nuclear membrane localization. Finally, removal of cardiolipin from the conjugating cells using inhibitor treatment showed that cardiolipin was required for the new macronucleus formation (including the expansion of macronuclear envelope) through the function of Drp6. Based on these results, authors concluded that cardiolipin targets Drp6 to the nuclear membrane in Tetrahymena.
The experimental data presented in this paper are reasonable and the results are solid, and therefore I think the deduced conclusions are convincing. However, to improve this paper, I have several minor comments to be revised before publication.
1) In the previous paper, it has been shown that GFP-Drp6 is localized in the inner nuclear membrane of both macronucleus and micronucleus. In this paper, however, this point is not clearly stated and is not shown in the figures. I could not understand such localization pattern of GFP-Drp6 in Figure 1C and Figure 3B and the statements in the text. I suggest adding such statements somewhere in Introduction or Result section. Also, add adequate references to the corresponding statements in the text.
We thank the reviewer for pointing out this important aspect of localization. We have included the statement in the Introduction section with appropriate reference.
2) Related to the comment 1, I suggest replacing Figure 1C (images of fixed cells) with Figure 1—figure supplement 1B (images of live cells) because nuclear localization of GFP-Drp6 are much clearer in Figure 1—figure supplement S1B (live cell) than Figure 1C (fixed cell), and because fixation may cause artificial redistribution of the proteins. Please add arrows in those figures to point out the position of micronucleus in those figures if necessary.
The reviewer is correct about artificial redistribution of proteins that may arise from fixation. We thank the reviewer for the suggestion of replacing the fixed images with the live cell images in the main figure. We have now included live cell images in the main figure, and fixed cell images in the Figure 1—figure supplement 1 is also included to visualize the DAPI stained nucleus.
3) Similarly, I suggest replacing images of Figure 5B (fixed cells) with those of Figure 3—figure supplement (live cells).
We have changed accordingly as suggested by the reviewer.
4) GFP-Nup3 is used as a marker protein of the nuclear pore complex (NPC). However, there is no description of how GFP-Nup3 is obtained or made. Add description how this DNA plasmid was obtained or generated.
We have now incorporated the information about how GFP-Nup3 plasmid DNA was obtained.
5) Related to the comment 4, "Nup3" is first discovered in Malone et al., 2009, but also soon after discovered as the name of "MicNup98B" in Iwamoto et al., 2009 and used in several papers including Iwamoto et al., Genes Cells, 2010; JCS, 2015; JCS 2017; and more. Because Nup3 is the Tetrahymena paralogs of human Nup98 and the name of "Nup98" is well established to call these homologs in various eukaryotes, I suggest adding the name of "MacNup98B" after the word of "Nup3" for reader's better understanding. I also suggest adding appropriate references to refer to this protein as follows: Add Malone et al., 2009 for "Nup3" and Iwamoto et al., 2009 for "MacNup98B."
We thank reviewer for pointing out and we now incorporated the name "MacNup98B" after the word "Nup3". We have also included the references suggested.
6) page 9, line 295: I wonder if "Figure 3B" may be a mistake of "Figure 5C." If so, please correct this.
We thank the reviewer for noticing this mistake. We have now corrected the figure number accordingly.
7) page 10, the second paragraph (lines 311-322): This paragraph discussed the possible involvement of Drp6 in the nuclear envelope expansion of the post-zygotic nucleus. It may be interesting to point out that large-scale nuclear envelope reorganization including the formation of the redundant nuclear envelope and the type-switching of the NPC (from the MIC-type NPC to the MAC-type one) has been reported at this developmental stage (Iwamoto et al., JCS 2015). For example, the peculiar shaped nuclear envelope with the redundant/overlapping nuclear envelope structure can be seen and the MAC-type NPCs rapidly assembles to the expanding nuclear envelope. It may be interesting to point out that cardiolipin and Drp6 may be involved in these phenomena. But it is too speculative and therefore consider adding such a discussion as an option.
We thank the reviewer for pointing out the correlation between cardiolipin and Drp6 with type switching of the NPC. As also mentioned by the reviewer, the correlation is highly speculative. Therefore, we have not added this in the Discussion.
8) Is the word "GFP-drp6-I553M" written in italics intended for the gene for the GFP-drp6-I553M protein? If so, protein may be acceptable here. Make sure there are no problems with italicized characters. Also, check if the lowercase letter "d" in "drp6" is OK because large letters are used in other cases.
As suggested by the reviewer, we have now changed "GFP-drp6-I553M" to “GFP-Drp6-I553M”.
9) Figure 1: I recommend switching the positions of HDyn1 and Drp6 in Figure 1A to keep the order in Figure 1B.
We have now switched the positions of HDyn1 and Drp6 in Figure 1A.
10) page 21, line 671: Add the word "Tetrahymena" before "Drp 6" to pair with the word "human dynamin 1".
Suggested change is incorporated
11) page 23, line 729: Remove "and."
Suggested change is incorporated
12) page 23, lines 729 and 731: Unify the expression of "cardiolipin" and "Cardiolipin"
Suggested change is incorporated
13) page 23, line 732: Add "or" before "10% Phosphatidylserin."
Suggested change is incorporated
14) Figure 3A: Please mark the position of I553M in the figure if possible. Alternatively, indicate the range of amino acid residues after the words "red" and "green" in the figure legend.
As suggested by the reviewer, we have now incorporated the positions of the amino acid residues in the figure legend
We thank the reviewer for the excellent comments that “the experimental data presented in this paper are reasonable and the results are solid, and therefore I think the deduced conclusions are convincing.” We also thank the reviewer for the minor comments which are thorough and very insightful. it improved the manuscript substantially. We have incorporated all the changes in the revised manuscript.
Reviewer #2 (Significance (Required)):
The corresponding author and his colleagues have reported that Tetrahymena Drp6 is localized to the outer nuclear membrane of both macronucleus and micronucleus of Tetrahymena (Elde et al., 2005) and that Drp6 is required for the formation of new macronuclei during nuclear differentiation (Rahaman et al., 2008). Therefore, these parts are not novel.
The novelty of this study is as follows:
1) The discovery of a specific amino acid residue (isoleucine at the 553rd) of Drp6 that is required for its nuclear membrane localization.
2) the discovery of a lipid molecule, cardiolipin, as a critical partner for Drp6's nuclear membrane targeting.
3) Discovery of involvement of cardiolipin in the new macronucleus formation (the expansion of macronuclear envelope) through the function of Drp6.
I think their findings are highly novel and will provide new insight into a field of cell biology. Especially, their findings will contribute to understanding how specific proteins targeted to the specific intracellular membranes. In addition, their methods (such as floatation assay) for analyzing the interaction between the protein of interest and lipid/liposomes will become an important tool.
We are very happy to note that the reviewer has pointed out the significance of the present study. We fully agree with reviewer and appreciate the thorough analysis and excellent conclusion from the reviewer.
[Editors' note: further revisions were suggested prior to acceptance, as described below.]
The additional request of one reviewer that should be addressed is to test the involvement of residue 552 and 554 to highlight the significance of isoleucine in position 553 in targeting Drp6 to cardiolipin.
We thank the reviewer for suggesting these excellent sets of experiments. We have now performed the cardiolipin binding and cellular localization of both the mutations at E552 and M554 residues. The results demonstrate that these two neighboring amino acid residues are not essential for providing specificity to bind cardiolipin and for localizing to nuclear envelope. These results clearly suggest that I553 is the key amino acid residue for conferring cardiolipin binding specificity and thereby specificity for nuclear recruitment. We have included these results in Figure 7.https://doi.org/10.7554/eLife.64416.sa2
- Abdur Rahaman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Prof. Aaron Turkewitz from the University of Chicago, Dr Kausik Chakraborty from IGIB, Prof. Jacek Gaertig from University of Georgia, Dr Utpal Nath from Indian Institute of Science, and Dr Renjith Mathew from National Institute of Science Education and Research for critical evaluation and useful comments on the manuscript. For other help, we thank members of this laboratory Sakti Ranjan Rout and Soham Mukhopadhyay. The work was partly funded by DBT grant (BT/PR14643/BRB/10/862/2010) to AR. The funders have no role in study design and interpretation, or the decision to submit the work for publication.
- Vivek Malhotra, The Barcelona Institute of Science and Technology, Spain
© 2021, Kar et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.
Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.