Together, the JMY and p53 depletion studies support the conclusion that JMY and p53 function in the same intrinsic apoptotic death pathway. The observation that HAP1 cells undergo apoptosis in a primarily pdependent manner, combined with the finding that JMY can associate with p53 and its acetyltransferase cofactor p [ 24 ], led us to next investigate the specific influence of JMY on p53 functions.
Since apoptosis is typically accompanied by p53 protein stabilization, post-translational modification, and re-localization into the nucleus [ 25 , 26 ], we examined these properties. Treatment of HAP1 and JMY KO cells with etoposide resulted in a clear increase in p53 protein levels, its phosphorylation on serine and serine, its acetylation on lysine, and its accumulation in the nucleus in the majority of cells S7 Fig , suggesting that JMY functions downstream of such pro-apoptotic changes in p53 stability and modification.
In response to genotoxic damage, nuclear p53 alters transcription and can trigger cell cycle arrest, DNA repair, apoptosis, senescence, and other stress responses [ 42 , 43 ]. For these experiments, we treated each cell line with DMSO or etoposide for 6h, removed the solvent or drug and replaced them with fresh media, and then quantified the numbers of total cells live and dead at regular intervals up to a 48h endpoint.
In contrast, when apoptotic cell quantities were measured using AnnV staining, the proportion of cells undergoing apoptosis was significantly higher in the HAP1 samples at every timepoint S8 Fig. Thus, without JMY, cells remain stuck in an arrested condition and fail to shift into a proper death signaling state.
One of the key p53 targets that triggers cell cycle arrest is CDKN1A , which encodes the cyclin-dependent kinase inhibitor, p21 [ 44 ]. Similarly, small amounts of p21 protein were found in HAP1 and JMY KO cells at steady-state, while treatment with etoposide resulted in a 3-fold increase in the abundance of nuclear p21 in both sets of cells S8 Fig. Collectively, these experiments suggest that JMY specifically promotes a cell suicide program and is not required for several other aspects of nuclear p53 modification or function, including the transcriptional responses that lead to a proliferation arrest.
Expression of the genes encoding p53, caspases, Bax and other Bclfamily members, additional key modulators of apoptosis, or JMY-interacting proteins such as p, Strap, and Mdm2 were not significantly different S10 Fig. Therefore, mutating JMY does not appear to adversely affect the basal expression of canonical apoptosis regulators or factors that are structurally or functionally related to JMY.
Each point represents the mean value from 3 independent RNA samples per genotype. Each point represents the mean value from two independent RNA samples per genotype, except for IL8 , which was only detected in one experiment. Since important differences in gene expression might not become evident until cells experience a pro-apoptotic stimulus, we next compared the transcriptomic changes that occurred in HAP1 and JMY KO cells after etoposide exposure.
Substantial changes in expression of genes that encode p53, caspases, Bcl-2 family members, or other prototypical apoptotic modulators were again not detected. Among the 19 genes that showed statistical differences between the parental and knockout cells, 16 displayed similar trends in either up- or down-regulation. These results imply that HAP1 and JMY KO cells are sufficiently equipped with enough potentially-apoptotic factors to be able to execute an intrinsic death program without a major increase in the abundance of pro-apoptotic transcripts.
Under the same genotoxic conditions, the BBC3 transcript, which encodes the p53 upregulated modulator of apoptosis PUMA, was increased 2. Therefore, mutating JMY does not appear to drastically affect the expression patterns of canonical p53 target genes, further suggesting that transcriptional reprogramming is not the principal driver of death signaling under the conditions used in our experiments.
In light of these observations, we sought to better understand how unanticipated factors might be contributing to cell survival or death in the presence and absence of JMY. Transcript levels for other small G-proteins were virtually the same between parental and KO cells S10 Fig , implying that the change for RHOD might be functionally meaningful. These experiments reveal that although the loss of JMY may not cause any obvious compensatory changes in the transcription of actin nucleation factors, it can result in increased expression of a G-protein known to affect cytoskeletal dynamics.
RhoD is a Rho-family GTPase that regulates many cellular functions, including actin assembly during filopodia formation, cell migration, endosome dynamics, and Golgi trafficking [ 45 — 56 ]. Additionally, RhoD appears to participate in other processes that affect cell proliferation, such as cell cycle regulation and cytokinesis [ 49 , 57 ].
Since JMY is involved in proteostasis and genotoxic stress responses through its activities in autophagy and apoptosis, we reasoned that the increase in RHOD expression in JMY KO cells might be part of a compensatory pro-survival mechanism that allows the cells to better tolerate the loss-of-function mutation in JMY.
This latter result supports the idea that RhoD normally promotes cell survival. Because of the complexities in studying RhoD activities during apoptosis in JMY KO cells, which appear to be fundamentally defective in their intrinsic apoptotic responses, we next wanted to characterize the effects of transiently depleting or permanently inactivating RHOD in otherwise healthy JMY-proficient cells.
Thus, decreasing RHOD expression can increase stress-induced apoptosis and necrosis when normal JMY and p53 responses are present, further strengthening the conclusion that RhoD plays a basic pro-survival role in cells.
Significance stars refer to comparisons of siControl vs siRhoD samples. Nucleotide is predicted to be absent in mRNA from both knockouts, whereas nucleotide should be present in both, albeit reflecting transcript levels that are lower than the parental control due to nonsense-mediated decay. Bands from this gel are also shown in panel A. Representative images and immunoblots appear in S11 Fig.
Additionally, in dose-response experiments, at each etoposide concentration the RhoD KO cell lines showed a death frequency that was approximately twice that of parental cells Fig 7H.
These studies reveal that the permanent loss of RhoD makes cells more susceptible to dying during normal culture conditions and also enhances their apoptotic responses following acute DNA damage. Since JMY and WHAMM are necessary for an efficient pathway of cyto c release, initiator caspase cleavage, and executioner caspase activation, we tested if RHOD inactivation affected these aspects of intrinsic apoptosis.
Following 3h and 6h of etoposide treatment, compared to the eHAP parental cell line, the RhoD KO samples had significantly higher percentages of cells with diffuse cyto c localization, greater levels of initiator caspase-9 processing, more cells undergoing cleavage of DEVD-containing substrates, and more cells staining positive for active caspase-3 Figs 7I—7K and S Arrowheads highlight examples of cleaved caspasepositive clusters.
G For quantification of E , the cleaved caspase-3 band intensities were normalized to the loading control intensities and the vector sample was set to 1. Compared to the vector-transfected knockout cells, which contained low levels of cleaved caspase-3, cells transfected with the wild type JMY construct showed a 4. In contrast, the JMY mutant with the WWW segment deleted failed to rescue apoptosis in the knockout cells, as cleaved caspase-3 levels were indistinguishable from those of the vector-transfected controls Fig 8E and 8G.
JMY is primarily cytoplasmic in healthy cells [ 27 , 28 ], and is typically redistributed into both the cytoplasm and nucleus upon exposure to apoptosis-inducing stressors [ 29 , 30 ]. Further, staining with fluorescent phalloidin demonstrated that filamentous- F- actin also assembled throughout the caspase-3 region and was most intense adjacent to JMY Fig 8F.
These findings highlight important relationships between JMY-mediated actin polymerization and the formation of cytoplasmic territories containing active caspase Because caspase-3 activation is driven by apoptosomes, macromolecular platforms consisting of cyto c , procaspase-9, and other scaffolding factors [ 38 , 61 , 62 ], we revisited the potential connections among JMY, cyto c , and actin in the cytoplasm.
They were also associated with bright clouds of the cleaved DEVD reporter prior to its nuclear import , indicative of active caspase-3 being present in proximity to these JMY- and cyto c -containing clusters Fig 9E.
While the fraction of parental cells harboring cyto c puncta increased during 3h and 6h exposures to etoposide, the JMY KO lines did not form cytosolic cyto c puncta under any of these conditions Fig 9F. F-actin appeared to be specifically reorganized, rather than globally polymerized or upregulated, as the inactivation of JMY did not significantly affect the overall levels of actin filaments, stained with phalloidin, or the total amounts of actin in the cytoplasm or nucleus, stained with an actin antibody S12 Fig.
These observations are consistent with a role for cytoplasmic JMY and F-actin during the period of apoptosis encompassing apoptosome assembly and executioner caspase-3 activation. Arrowheads and magnifications i-iv highlight examples of cytosolic JMY puncta. Significance stars refer to comparisons between DMSO and etoposide treatments. Arrowheads and magnifications i,ii show examples of cytosolic JMY and cyto c puncta.
G U2OS cells were treated with etoposide, fixed, and stained with antibodies to JMY green and cyto c magenta , and with phalloidin F-actin; yellow. Magnifications i depict clusters of JMY, cyto c , and F-actin.
These phenotypic differences are reminiscent of the reduced versus increased apoptotic characteristics previously observed in the WHAMM and RhoD knockout cells, respectively. Additionally, this dominant interfering RhoD protein increased JMY levels in the nucleus Fig 10C—10E , signifying that the nucleotide-bound state of RhoD affects both the formation of the punctate cytosolic structures and the nuclear accumulation of JMY.
Collectively, these results indicate that although JMY and RhoD exhibit opposing pro-apoptotic versus pro-survival activities, both proteins apparently function in the assembly and organization of cytoplasmic territories containing cyto c , active caspases, and F-actin. Arrowheads highlight examples of cytosolic JMY puncta. Significance stars refer to comparisons to parental cell lines.
GFP is shown in magenta. Magnifications i-iii represent examples of JMY localization. The polymerization, organization, and turnover of actin filaments have been thoroughly characterized during many cellular functions that maintain viability [ 9 , 10 ].
However, the contributions of actin assembly factors to apoptotic cell death are not well understood. Among the best-known cytoskeletal features of apoptosis are the actin filament rearrangements and disassembly that accompany changes in cell adherence and morphology [ 7 , 8 ].
Actin itself can be cleaved by caspases [ 63 , 64 ], resulting in non-polymerizable fragments that promote apoptotic cellular phenotypes [ 65 — 67 ]. The actin turnover machinery has also been implicated in controlling apoptosis at multiple steps. Cofilin, which depolymerizes actin filaments [ 68 ], influences the early stages of apoptosis, as it translocates to mitochondria and may increase the release of cytochrome c [ 69 — 71 ] and the localization of p53 [ 72 ].
Actin is additionally recruited to mitochondria at around the time of mitochondrial permeabilization [ 73 — 75 ]. During the later stages of apoptosis, the F-actin-severing protein gelsolin [ 76 ] is cleaved by caspases, resulting in a fragment with unregulated severing activity that drives depolymerization [ 77 ] and allows chromatin fragmentation [ 78 ].
In contrast to these described roles for actin disassembly proteins in apoptosis, less is known about the actions of the actin assembly machinery during cell death. WASP-family proteins have many well-recognized activities, but the extent to which they participate in cell death pathways is relatively underexplored.
WAVE1 has been one of the most studied members, as it can affect the localization or modification of Bclfamily proteins. Several transient overexpression and depletion experiments suggest that WAVE1 limits mitochondrial permeabilization [ 20 — 23 ]. But in our initial knockout screen using BCR-ABL-immortalized human cells, the permanent loss of WAVE1 did not cause a consistent increase in the frequency of apoptosis, so the degree to which WAVE1 acts as an anti-apoptotic factor in a cell type specific manner requires further investigation.
JMY was discovered as a cofactor that enhanced the transcriptional activity of p53 [ 24 ], and subsequent RNAi studies indicated that JMY has pro-survival [ 32 ] or pro-death [ 27 , 29 , 79 ] functions under different experimental circumstances. Our work advances the understanding of its pro-death role by demonstrating that cells with permanent deletions or transient depletions of JMY exhibit significant defects in apoptotic processes following DNA damage.
WHAMM did not have any previously described roles in apoptosis, so the latter observation adds a third WASP-family member to the repertoire of actin assembly factors that can function in programmed cell death. Although the two proteins participate in similar cellular activities, they exhibit some key molecular differences. WHAMM localizes to the ER and cis -Golgi and binds microtubules to mediate membrane tubulation and transport [ 37 , 47 , 83 ], while JMY acts later in the secretory pathway to promote trafficking away from the trans -Golgi [ 80 ].
JMY binds to LC3 and polymerizes actin at autophagosomes during their maturation [ 32 ] and actin-based motility [ 28 ]. While it is reasonable to assume that the functions of WHAMM and JMY in secretion and autophagy help sustain cells during normal growth and allow them to adapt to conditions of nutritional stress, the fact that both factors also enable cell death indicates that they function as pivotal players in the cellular responses to multiple other stressors including genotoxic insults.
In contrast, JMY or WHAMM knockout cells showed significant reductions in the terminal apoptosis phenotypes of phosphatidylserine externalization and membrane permeabilization. These results indicate that both cytoskeletal regulators are key participants in a rapid, intrinsic, pdependent cell death pathway, with JMY acting as the more prominent contributor.
Under normal cell culture conditions, JMY is found predominantly in the cytosol [ 27 , 28 ], but in response to DNA damage, it accumulates in the nucleus while still maintaining a cytosolic presence [ 29 , 30 ]. At steady-state, WHAMM is mostly associated with cytoplasmic membrane-bound organelles, although it displays a nuclear localization in a small subpopulation of cells [ 37 ]. Such observations are consistent with the existence of both cytoplasmic and nuclear roles for these proteins in apoptosis.
JMY was previously shown to interact with the stress-response protein Strap, the acetyltransferase p, and p53 [ 24 , 31 ]. However, our current work suggests that the activity of JMY in transcriptional regulation via p53 may not be the primary apoptotic driver following DNA damage. In our experiments, etoposide-induced increases in the overall abundance, nuclear accumulation, phosphorylation, and acetylation of p53 occurred in the presence or absence of JMY.
Moreover, JMY inactivation had little effect on transcriptional upregulation of the p53 target CDKN1A , which encodes the key cell cycle inhibitor p In fact, JMY-deficient cells exhibited a prolonged arrest in response to DNA damage, indicating that JMY is not necessary for the gene expression changes that stop proliferation.
These results collectively support the idea that JMY participates in only a subset of the pmediated transcriptional responses to genotoxic stress. Given the speed and efficiency with which p and JMY-proficient parental cells underwent apoptosis, we hypothesized that they constitutively express a pool of potentially apoptotic factors that enable the cells to respond rapidly to DNA damage. Indeed, global and targeted gene expression profiling experiments implied that inactivation of JMY did not adversely affect the expression of common apoptosis regulators either before or after etoposide exposure.
RhoD has wide-ranging functions in regulating actin dynamics at the plasma membrane, endosomes, and Golgi, and in influencing cell proliferation [ 45 — 57 ]. While the selective pressures that led to greater RHOD expression in two out of the three JMY knockout cell lines are unknown, we nevertheless reasoned that increased RhoD levels may be part of a compensatory pro-survival mechanism that allows cells to better tolerate a permanent JMY mutation.
Consistent with this possibility, RhoD depletion or deletion experiments resulted in more necrosis and enhanced apoptotic responses—the opposite phenotype of JMY- or WHAMM-depleted cells. These findings reveal complex relationships among two actin nucleation factors and one small G-protein during cell death and survival S12 Fig. Most notably, wild type and actin polymerization-deficient mutants of JMY were both able to congregate into discrete cytosolic structures, but only the polymerization-proficient protein generated distinct juxtanuclear zones containing actin filaments, active caspase-3, and cytochrome c.
In light of these observations, we propose that JMY-mediated actin assembly is required for creating cytoplasmic territories that direct the activation of executioner caspase-3 by cytochrome c -containing apoptosomes S12 Fig. The structural and biophysical properties of apoptosomes have begun to come into focus [ 38 , 61 , 92 ], yet their cell biological characteristics remain less clear.
Continuing to define the roles of different elements of the actin assembly and regulatory machinery in cellular adaptations to endogenous or exogenous stress could provide new avenues for understanding tumorigenesis. Similarly, the functions of WHAMM and JMY in autophagy [ 32 , 81 , 82 ] could enable cancer cells to better survive in diverse physiological environments and in response to chemotherapy [ 95 , 96 ]. However, our current results describing apoptotic requirements for JMY and WHAMM support the idea that these factors also possess key tumor-suppressive features.
TP53 is well known as the most commonly mutated gene in human cancers, and mutations that inactivate p53 or other passociated proteins are linked to poor prognoses [ 97 — 99 ]. Interestingly, JMY expression also appears to be lost in several B-cell lymphomas and invasive carcinomas [ ]. Given the new positions of JMY and WHAMM as important players in pdependent apoptotic pathways, a greater understanding of how their actin nucleation, proto-oncogenic, and tumor-suppressive activities are coordinated will likely shed further light on how programmed cell death mechanisms are impacted by the cytoskeleton.
This study did not include research with human subjects or live animals. Human cell lines were acquired from Horizon Genomics or the UC Berkeley cell culture facility as described below. HAP1 cells are nearly-haploid fibroblast-like cells that contain an immortalizing BCR-ABL fusion and a single copy of all chromosomes except for a heterozygous 30Mb fragment of chromosome 15, which is integrated within the long arm of chromosome 19 [ 33 , ].
All assays were performed using cells that had been in active culture for 2—10 trypsinized passages. Plasmids were maintained in E. Surviving colonies were collected, expanded in well plates, 6-well plates, and ultimately T flasks prior to cryopreservation.
Upon re-animation, G concentrations were reduced stepwise to 1. For transient expression, U2OS cells were transfected in 6-well plates with ng of DNA prior to reseeding onto 12mm glass coverslips in well plates and fixing as described below.
Transfection increased cell death in Fig Equivalent volumes of DMSO were used as controls. Cells cultured in 6-well plates were collected and processed for immunoblotting or RT-PCR, and cells cultured in well plates were used in live fluorescence microscopy assays.
After 24h of growth, cells were subjected to control-, DMSO-, or etoposide-containing media changes and rinsed with phosphate buffered saline PBS. The data summarized in this publication can be found in Supporting Information described below. Detached cells were collected from 6-well plates and combined with adherent cells in PBS containing EDTA, centrifuged, washed with PBS, and centrifuged again to ensure collection of all live and dead material.
For live fluorescence-based assays, approximately 2. For mitochondrial visualization in Fig 9 , MitoTracker Red Invitrogen was added for 30min prior to fixation. For immunofluorescence microscopy, approximately 2. Cells were probed with primary antibodies S3 Table diluted in blocking buffer for 45min. All cells were viewed in multiple focal planes, and Z-series were captured at 0. Images presented in the figures represent one slice or two-slice projections. For analyses of total cellular p53, JMY, or actin levels, the Selection tool was used in the phalloidin channel to select individual cells, and the Measure tool was used in the p53, JMY, or actin channel to measure the mean fluorescence intensity per cell.
For analyses of cytoplasmic JMY or actin levels, the nuclear intensity was subtracted from the total intensity for individual cells. These results indicate that, immediately after mixing salts, the repulsive force between the molecules disappears, while almost all of actin molecules still remain in the monomeric state. Each plot is derived from the intensities integrated between 0.
The SAXS intensities are corrected by the transmission of each buffer. Each I 0 was derived from the y -intercept of the extrapolated straight line of a Guinier plot of each I q.
Actin initiates polymerization shortly after the disappearance of the inner intensity fall. The curve appeared to be expressed as a cubic function of time with a downward convex shape, ensuring that the actin polymerization under the present conditions is in accordance with a nucleation-controlled reaction scheme 19 see Supplementary note 2. Figure 3a shows the time-resolved SAXS intensity profiles during salt-induced polymerization of actin.
The data were taken from a. For details, see text. The existence of the isoscattering point suggests that the polymerization of actin may be described as a two-state transition between G- and F-actin without an intermediate phase. However, when singular value decomposition SVD analysis 30 see Methods and Supplemental note 3 was applied to the time-resolved SAXS data, we found that there exist three components that contribute significantly to the scattering intensities.
Mallnowski proposed the indicator IND function for the determination of the number of factors included in the data from the SVD The dimension giving the minimum of the IND function is 3. This value exhibits the number of factors sufficient for the description of the SAXS data, corresponding to the minimum number of the kinds of complexes existing in the process of polymerization.
Therefore, there is a third complex, other than G- and F-actin, during the polymerization. The SAXS profiles are described by scattering terms from individual actin molecules constituting oligomers as well as monomers in solution and interference terms between the actin molecules in oligomers At such an isoscattering point, the interference terms for all oligomer species larger than the trimer should be zero see Supplementary note 4.
This suggests that the oligomers formed during actin polymerization share the same structural periodicity as in the F-actin filament. Therefore it would be sufficient to postulate F-actin-like helical oligomers for scattering analysis of polymerization process.
For the following analysis of time-resolved SAXS data, helical oligomers consisting of 3—7 molecules in addition to monomers and dimers were assumed to be involved as the components existing during early polymerization. Two kinds of dimers, those with a parallel configuration Supplementary Figure 2a and an anti-parallel configuration Supplementary Figure 2a , were taken into consideration.
Such a dimer with an anti-parallel configuration has frequently been detected at the early stage of polymerization of actin 32 , 33 despite the fact that this configuration does not exist in normal F-actin. The scattering profiles of these two dimers are distinctive. The scattering intensity profiles calculated for the constructed oligomer models together with these dimer models are depicted in Supplementary Figure 3 and employed as the theoretical intensity profiles. For all of possible combinations of the species, each time-resolved SAXS intensity profile was fit to a weighted sum of the theoretical profiles of the species, from which the results the AIC were calculated.
Figure 4 shows the frequencies of species combinations that were selected for the intensity profiles in each s time division. Collectively, the anti-parallel dimer is formed at the initial stage after mixing salts, and F-actin appears thereafter. Therefore, third dominant complex present during early polymerization is the anti-parallel dimers.
The histograms were made from 16 time-series of the SAXS intensity profiles during polymerization. The marks of the species combination express as follows: G G-actin , 2A anti-parallel dimer , 2P parallel dimer , 3 trimer , 4 tetramer , 5 pentamer , 6 hexamer , 7 heptamer and F F-actin. As another example of the weight fractions obtained in the AIC analysis, Fig.
Characteristics of the weight fractions are consistent with the description deduced from the above minimum preferred model. In addition, the oligomers other than the anti-parallel and parallel dimers, and heptamer do not appreciably accumulate at the early stage of polymerization. This is supportive evidence that the actin polymerization under the present conditions is a nucleation-controlled reaction The bars express the weight fractions for oligomer species [G-actin G , dimers 2A and 2P , trimer 3 to heptamer 7 and F-actin F ] with error bars of the standard deviations for species.
We set up the following kinetic model including the formation of anti-parallel non-polymerizable dimers A s , in which actin molecules A polymerize in sequence from G- to F-actin 35 , 36 , see Supplementary note 5. In the above reaction scheme, it is generally assumed that once the oligomer i gets to be large, the rate constants for elongation become that for F-actin regardless of the size i 16 , With this parameter set, the time-resolved SAXS intensity profiles during the early polymerization in series of It is worth mentioning that the observed data could not be fitted well by the reaction scheme without including the non-polymerizable dimers.
We derived the time-course of the weight fractions of species existing during the early polymerization together with the fraction of monomers and the result is shown in Fig.
It turned out that the anti-parallel dimer is dominantly formed at the earliest stage just after mixing salts, and F-actin appears thereafter. The trends were mostly consistent with the result of the AIC analysis described above. G-actin solid , non-polymerizable, anti-parallel dimer dash , and F-actin dash-dot are major components.
A small amount of polymerizable, parallel dimer dot is also observed. The amounts of the other oligomers are nearly zero. The diagram corresponds to that of the apparent free energy change below in presence of 1M actin. The type of diagram is a nucleation-controlled polymerization in the category of cooperative polymerization The nucleus is a tetramer corresponding to a maximum in the free energy diagram.
The diagram is compared with those in the previous studies 16 , 18 , 36 , 40 in Supplementary Figure 4. The reactions before the tetramer formation correspond to an unfavorable nucleation while those after the tetramer formation to a favorable elongation. This type of polymerization has been classified as a nucleated cooperative polymerization 20 , where the oligomer with the highest free energy change is a nucleus.
Figure 6c shows that it is the tetramer. Steady elongation starts at the pentamer with the same rate constants for monomer addition to F-actin. The time-resolved SAXS intensities during salt-induced polymerization of actin appear to have the apparent isoscattering point Fig.
However, the diagram of the free energy change of oligomer formation Fig. According to the definition of Ferrone 19 , 20 , the nucleus of the reaction is a helical tetramer because it is the least stable intermediate oligomer with respect to the monomer and exists only transiently in the polymerization process Fig.
This conclusion is consistent with the previous, kinetic studies of the actin polymerization by Tobacman and Korn 15 and by Fesce et al. In parallel to the nucleation, non-polymerizable dimers are formed accumulated during the early polymerization Figs 4 and 6a. The size of the nucleus in actin polymerization has been discussed in terms of the simple geometrical explanations 43 , As discussed in Supplementary note 6 , the formation of a dimer or a trimer as a nucleus has been deduced from the structural geometry of F-actin However, we showed here that the nucleus for polymerization of actin is a tetramer.
The nucleus size is probably related to the structure of the end receiving a monomer. As shown in Fig. The implication for this is that end of the trimer has an incomplete structure see Supplementary Figure 5c.
By contrast, in Fig. This suggests that the structure of the end receiving an additional monomer in the tetramer would be identical to that of the fast growing B-end of F-actin, probably having the same subunit disposition as in F-actin.
The subunit disposition would require the flattening of actin molecule that was found upon the association of G-actin to F-actin filament This is because the flattening is considered to be necessary for generating the canonical subunit arrangements in F-actin The flattening is produced by a relative rotation of two main domains constituting an actin molecule around the line passing through subdomains 1 and 3, being stabilized by forming the longitudinal contact with the other subunit at subdomains 2 and 4 45 see Supplementary Figure 5b.
We deduce that two subunits at the B-end of tetramer, colored by red in Supplementary Figure 5c , have a flat conformation, and that the diagonal contacts between the two subunits can generate the canonical disposition like the B-end of F-actin, serving as a nucleus for further polymerization. More detailed discussion based upon crystal structures of actin oligomers will be presented elsewhere. In Fig. The addition of neutral salts diminishes the electrostatic repulsion force between G-actin molecules in G-buffer, allowing them to closely approach each other.
Activation of monomer via salt binding which is thought to be prerequisite for initiation of polymerization 40 , 43 , 47 would simultaneously occur, though it could not be detected by the SAXS at the present resolution. The addition of salts engenders a large excess of monomers capable of polymerization through a decrease in the critical concentration the concentration of monomer in equilibrium with polymer of actin for polymerization, typically from ca.
Then the dimeric molecules, such as parallel and anti-parallel dimers, are formed prior to a major nucleation. Dimers with an anti-parallel orientation of subunits do not contribute to the nucleation, though they are thought to be incorporated transiently into growing filaments 32 , Meanwhile, from the polymerizable dimers, the trimer and the tetramer are sequentially formed, though these are energetically unfavorable reactions.
Once monomers are added to the tetramers, the reaction proceeds along the free energy downhill slope, and F-actin filaments start to grow. Although the formation of nuclei ceases with a decrease in polymerizable monomers, the growth of filaments continues until the concentration of monomers comes to reach the critical concentration for polymerization, resulting in the accumulation of long F-actin filaments. Nucleation process includes the formation of dimers, trimer, and tetramer, and followed by canonical growth process is from the tetramer.
For rapid generation of new filaments in cells, it would be essential to generate the end structure with a diagonal disposition of subunits similar to the B-end of F-actin and to keep the disposition until the subsequent monomers are added. Two actin molecules help the other two molecules to form the diagonal disposition probably via the promotion of flattening in the case of actin alone.
By contrast, in cells, nucleators having two or more binding sites to actin molecules, promote a formation of such a diagonal disposition.
The longitudinal contact might maintain the actin subunit in the flat conformation, promoting the formation of nucleus. However, the recruitment and delivery of the subsequent monomer are still left behind.
Recently, an interesting model for Vop-L has been proposed by Zahm et al. Actin was prepared from chicken breast muscle according to the method described previously Actin was furthermore purified by size-exclusion chromatography with a HiLoad Superdex column GE healthcare. The actin thus obtained was used as Ca-actin. The 1D-intensity profiles after averaging for the buffer solution measured under the same experimental conditions were subtracted from those for the actin solution.
The operation of the stopped flow mixer was synchronized with the data-acquisition system. The measurement was repeated with a different time delay to obtain data series reflecting the time sequence of polymerization.
After checking the lack of any sample damage effects by the X-ray exposure for each pattern, a series of the intensity profiles with the same time delay were averaged to obtain one profile.
To empirically decide the minimum number of oligomeric components, we calculated the indicator IND function that was proposed by Mallnowski The number dimension giving the lowest value of the IND function is the substantial rank of the matrix see Supplementary note 3. Coordinates for the anti-parallel dimer were made from the arrangement of the anti-parallel dimer in the actin crystal of PDB code: 1LCU 59 , and those for the parallel dimer were made from the arrangement along the crystal contacts of PDB code: 2FXU Coordinates for F-actin-like helical oligomers were prepared from the F-actin model by Oda et al.
The model intensity profiles of the dimers and the trimer were scaled against that of the tetramer using their I 0 values. We calculated the averages and their deviations of intensities from the selected profiles at each scattering vector length q. One hundred averaged profiles with the deviations were prepared from one measurement. We treated one hundred SAXS intensity data not as one averaged data to reduce the effect of random errors included in each measurement on deconvolution.
These intensity profiles were deconvoluted by all sets of possible combinations of the model profiles obtained from G-actin, anti-parallel dimers, parallel dimers, trimers, tetramers, pentamers, hexamers, heptamers and F-actin. We adopted the combination of the species giving the lowest AIC value as a preferred combination for each averaged profile. The procedure was performed for 16 independent time-series of the SAXS intensity profiles that were measured under the same experimental conditions.
Plausible rate constants were determined by fitting the SAXS intensity profiles that were computed using the kinetic scheme of polymerization see Supplementary note 5 to the observed time-resolved SAXS intensity profiles with actin concentrations of Second, under the condition of the minimal i 0 , the initial sets of parameters were systematically, randomly generated, from which the plausible sets of the rate constants were searched for.
We prepared initial sets. How to cite this article : Oda, T. Early nucleation events in the polymerization of actin, probed by time-resolved small-angle x-ray scattering.
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Zhang, Z. Rice morphology determinant encodes the type II formin FH5 and regulates rice morphogenesis. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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