F- homeostasis through transcriptional regulation and proteasome-mediated proteolysis

Masayuki Onishia, Kresti Pecanib, Taylor Jones IVa, John R. Pringlea,1, and Frederick R. Crossb,1

aDepartment of Genetics, Stanford University School of Medicine, Stanford, CA 94305; and bThe Rockefeller University, New York, NY 10065

Contributed by John R. Pringle, May 21, 2018 (sent for review December 26, 2017; reviewed by Susan K. Dutcher and Daniel J. Lew)

Many organisms possess multiple and often divergent different actin isoforms. Its genome encodes two actins, the con- whose regulation and roles are not understood in detail. For ventional IDA5 (∼90% identical to vertebrate actins) and the un- example, Chlamydomonas reinhardtii has both a conventional actin conventional NAP1 (∼65% identical to IDA5; refs. 15 and 16). (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in Vegetative wild-type cells express only IDA5, which forms filaments normal proliferating cells. We showed previously that the drug latrun- that localize to the cell cortex, around the basal bodies, and in cage- – culin B (LatB) causes loss of filamentous (F-) IDA5 and strong up- like structures around the nucleus (17 20). NAP1 is expressed regulation of NAP1, which then provides essential actin function(s) transiently during mating and flagellar regeneration and localizes to the fertilization tubule, basal bodies, and flagella (19, 21), where by forming LatB-resistant F-NAP1. RNA-sequencing analyses now – show that this up-regulation of NAP1 reflects a broad transcriptional IDA5 is also present (17 19, 22, 23). NAP1 is also constitutively up- response, much of which depends on three (LAT1, LAT2, and regulated in ida5 null mutants (17). Despite its divergent primary sequence, NAP1 provides essential actin functions, as shown by the LAT3) identified previously as essential for NAP1 transcription. Many viability of ida5 null mutants and inviability of ida5 nap1 double of the LAT-regulated contain a putative cis-acting regulatory “ ” mutants (17, 20). site, the LRE motif. The LatB transcriptional program appears to be We reported previously that vegetative Chlamydomonas cells activated by loss of F-IDA5 and deactivated by formation of F-NAP1, lose F-actin (F-IDA5) within ∼10 min when treated with the drug – thus forming an F-actin dependent negative-feedback loop. Multiple latrunculin B (LatB) (19, 20), which blocks actin polymerization in genes encoding proteins of the ubiquitin-proteasome system are most organisms by binding strongly to G-actin subunits (24–26). among those induced by LatB, resulting in rapid degradation of

However, the LatB-treated cells up-regulate transcription of NAP1 CELL BIOLOGY IDA5 (but not NAP1). Our results suggest that IDA5 degradation is within 10–30 min, and NAP1 then forms filaments that are functionally important because nonpolymerizable LatB-bound IDA5 highly resistant to LatB (20). As a result, Chlamydomonas cells interferes with the formation of F-NAP1. The genes for the actin- continue to proliferate without a detectable pause after addi- interacting proteins cofilin and profilin are also induced. Cofilin induc- tion of LatB. A genetic screen for LatB-sensitive mutants tion may further the clearance of IDA5 by promoting the scission of F- yielded mutations in NAP1 and in three other genes, LAT1, IDA5, whereas profilin appears to function in protecting monomeric LAT2,andLAT3, whose products are all required for the induction IDA5 from degradation. This multifaceted regulatory system allows of NAP1 upon LatB treatment. LAT2 is a protein of unknown rapid and quantitative turnover of F-actin in response to cytoskeletal function for which homologs can be found only in closely related perturbations and probably also maintains F-actin homeostasis under green algae (Volvocale lineage), whereas LAT1 and LAT3 are normal growth conditions. predicted protein kinases with homology to p21-activated kinases

actin | algal cytoskeletons | Chlamydomonas | latrunculin | Significance proteasome Cytoskeletal actin microfilaments have roles in cell-shape de- ctin is one of the most highly conserved eukaryotic proteins termination, motility, membrane trafficking, and cell division. Aand plays important roles in a wide range of biological Actin filaments respond dynamically to environmental changes processes, including the determination of cell shape and polari- and shifting cellular needs, and functionally different actin zation, vesicle transport and endocytosis, cell motility, and cy- subtypes may play important roles in such responses. The alga tokinesis (1–4). Actin functions primarily in its filamentous form Chlamydomonas has two actins: IDA5, an actin of conventional (F-actin) rather than as globular monomers (G-actin). F-actin can sequence that is expressed in normal growing cells, and NAP1, be nucleated by either of two major nucleators: formins, which a divergent actin that is normally not expressed. Disruption of nucleate the formation of long, unbranched filaments and bundles, IDA5 filaments results in rapid transcriptional induction of and Arp2/3 complexes, which induce the formation of branched NAP1 and hundreds of other genes, rapidly replacing all meshworks (5). Different species contain different numbers of IDA5 filaments with NAP1 filaments, in part by proteasome- genes encoding actins, ranging from 1 in yeast and Giardia (6–8), mediated degradation of IDA5. This system allows resistance Chlamydomonas through 6 in vertebrates (9), 2 to 21 in land plants (10, 11), of to actin-depolymerizing drugs and proba- and >30 in the slime mold Dictyostelium discoideum (12). When bly also compensates for other, diverse actin cytoskeletal per- multiple actin genes are present, they are often transcribed in turbations, whether intrinsic or induced. distinct patterns under different environmental conditions and/or Author contributions: M.O., K.P., T.J., J.R.P., and F.R.C. designed research; M.O., K.P., T.J., at different stages of development and in different cell types of and F.R.C. performed research; M.O., K.P., T.J., J.R.P., and F.R.C. analyzed data; and M.O., multicellular organisms. In most organisms, it is not known how J.R.P., and F.R.C. wrote the paper. the different actin isoforms are regulated and contribute differ- Reviewers: S.K.D., Washington University in St. Louis; and D.J.L., Duke University entially to cellular processes because of the challenges posed for Medical Center. genetic and molecular analyses by their high degree of structural The authors declare no conflict of interest. similarity and functional redundancy. In a few cases, there is good Published under the PNAS license. evidence that actin isoforms have functional differences (9, 13, 14), 1To whom correspondence may be addressed. Email: [email protected] or fcross@ but even in these cases it is not fully understood how the abun- mail.rockefeller.edu. dance of the different isoforms is regulated. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. The unicellular green alga Chlamydomonas reinhardtii provides 1073/pnas.1721935115/-/DCSupplemental. an attractive system for study of the regulation and function of

www.pnas.org/cgi/doi/10.1073/pnas.1721935115 PNAS Latest Articles | 1of10 Downloaded by guest on September 24, 2021 and MAPKKKs (20). Such kinases are conserved widely in eukary- seq) to analyze the transcriptome of LatB-treated wild-type cells otes, and some of them have been implicated in cellular responses to through a time course of 120 min (Materials and Methods, RNA- perturbations of F-actin (27–29). Taken together, the results sug- seq Experiment 1). An ida5-1 null mutant (15, 20) was included gested that Chlamydomonas has a pathway that monitors the integrity as a control because this strain neither shows an obvious cellular of the F-actin cytoskeleton and maintains its homeostasis. response nor induces NAP1 in response to LatB treatment (20). Our previous study (20) left several major questions open. After normalization of read counts (Materials and Methods), First, how does the actin-homeostasis pathway sense the per- k-means clustering was used to group the genes into six clusters turbation in F-actin? Second, what other factors are involved in based on the similarity of their expression profiles (Fig. 1A). the pathway? Third, does the pathway induce genes in addition Clusters 1 (2,063 genes) and 5 (2,216 genes) showed significant to NAP1 that are also important for the ability of Chlamydo- induction and repression, respectively, in the presence of LatB; monas cells to continue proliferating in the presence of LatB? NAP1 was a conspicuous member of cluster 1 (Fig. 1B, top line). Here, we used transcriptomic and genetic analyses to explore The genes in these clusters were largely unresponsive to LatB in these questions further. the ida5-1 mutant (Fig. 1A), indicating that the transcriptional responses were specifically dependent on the LatB–IDA5 interaction. Results In contrast, the genes in clusters 2, 3, 4 (Fig. 1A), and 6 (see Fig. 1A Global Transcriptional Response to LatB. To identify genes that are legend) showed only small changes, and/or changes that were similar regulated in response to LatB, we used RNA sequencing (RNA- in wild-type and ida5-1 strains, in response to LatB. Thus, LatB

Log fold-change from WT at t=0: ABExpression changes (all genes) 2 −10−5 0 5 10 Time WT + 10 μM LatB ida5-1 + 10 μM LatB Expression changes (actin-cytoskeleton genes) (min):0 3 10 30 60 90120 0 3 10 30 60 90120 ida5-1 Time WT + 10 μM LatB + 10 μM LatB (min): 0 3 10 30 60 90120 0 3 10 30 60 90120 Cre03.g176833 NAP1 1 Cre07.g339050 COF1 Cre10.g427250 PRF1 Cre12.g545000 ARP7 Cre06.g249300 ARP5 Cre08.g382590 ARP6 Cre09.g416250 MYO2 2 Cre16.g658650 MYO1 Cre04.g229163 Formin Cre13.g603700 IDA5 Cre16.g648100 ARPC1A Cre05.g232900 Formin Cre16.g687500 ARP2 Cre13.g563800 MYO3 3 Cre09.g397950 ARPC3 Cre06.g249200 ARP4 Cre06.g311250 Formin Cre03.g166700 FOR1 Numbers of genes induced by LatB C (Fold-change ≥ 2, Padj < 0.05) 4 in WT in nap1 13 350 802

427 5 11 833

508 in lat3 D E 222 genes upregulated in WT Genes induced or repressed by LatB in WT (Fold-change ≥ 4, Padj < 0.05) WT ida5 lat1 lat2 lat3 nap1 0.6 LatB: –+–+–+–+–+–+ 0.4

Density 0.2

0 −2 0 2 4 6 8 35 genes downregulated in WT 1.0 0.8 0.6 0.4 Density 0.2 0 −6 −4 −2 0 2

Log2 fold-change (strain+LatB 90 min / WT at t=0)

Fig. 1. LAT-pathway-dependent transcription of a large but specific set of Chlamydomonas genes in response to depolymerization of F-actin by LatB. (A) Heat-map display of expression in wild-type and ida5-1 cells treated with LatB (see Materials and Methods, RNA-seq Experiment 1). All genes were grouped into six clusters based on similarity of expression patterns (k-means clustering using the data from both the wild-type and ida5-1 strains), followed by

a hierarchical sorting within each cluster. Log2 fold changes in read counts relative to wild type at 0 min are shown for each gene in clusters 1–5, which contain 2,063, 1,265, 3,114, 1,569, and 2,216 genes, respectively. The 7,514 genes in cluster 6 all showed little or no change in expression in either strain, so this cluster is omitted from the figure for simplicity. (B) Expression of actin-cytoskeleton genes in LatB-treated cells. Data for genes encoding actins, actin-related proteins (ARPs), formins, Arp2/3 complex subunits (ARPCs), myosins, profilin, and cofilin were extracted from A.(C and D) Expression in mutants of genes that were differentially regulated in wild type during a 90-min exposure to LatB (see Materials and Methods, RNA-seq Experiment 3). (C) Venn diagram showing the

numbers of genes up-regulated ≥twofold in the indicated strains (PADJ < 0.05). The “LAT-target” genes used for GO-term analysis (see text) are highlighted in yellow. (D) Heat-map display of gene expression for the 222 and 35 genes that were up-regulated or down-regulated ≥fourfold (PADJ < 0.05), respectively. (E) Smoothened density histograms of the expression changes for the up-regulated (Top) and down-regulated (Bottom) genes from D. Shown are the log2 fold changes of the genes in the indicated strains after LatB treatment relative to wild type before LatB treatment.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1721935115 Onishi et al. Downloaded by guest on September 24, 2021 causes changes in the expression of large but specific sets of genes in To ask if preexisting NAP1 protein is sufficient to activate the Chlamydomonas, in addition to NAP1. negative-feedback function and thus reduce or eliminate the The transcriptional response to LatB was almost completely initial transcriptional response to LatB, we used a strain in which distinct from the responses to the stress-inducing treatments of NAP1 is transcribed from the constitutively active TUB2 pro- pH shock (which is known to induce NAP1; ref. 21) and heat moter. However, the onset and subsequent attenuation of the stress at 33 °C (SI Appendix, Fig. S1A, Left and Center). In con- transcriptional response were essentially the same in this strain trast, we observed some overlap between the response to LatB as in wild type (SI Appendix, Fig. S2B). Perhaps IDA5 is pre- and that to translational inhibition with cycloheximide (CHX) ferred to NAP1 for forming filaments in the strain expressing (SI Appendix, Fig. S1A, Right), perhaps reflecting a nonspecific both proteins, and it is the assembly of F-NAP1 filaments, rather stress component to the LatB response. Alternatively, the over- than merely the presence of NAP1 protein, that is the key ele- lap may reflect an effect of CHX in compromising F-actin ment for the negative-feedback loop. structure or function, as has been observed in other organisms In contrast to the up-regulated genes, most of the 35 genes (30). When the 137 genes with the greatest differences in ex- down-regulated ≥fourfold were also repressed in the lat1-lat3 pression between the LatB-alone samples and the CHX-alone and nap1 mutants (Fig. 1D), suggesting that they are not tar- samples were examined, 40 genes were observed to be up- gets of the LAT pathway. regulated specifically by LatB and not CHX (SI Appendix, Fig. S1B). In most cases, this up-regulation was partially or entirely LAT-Response Element. Using the sets of genes that are differen- suppressed when CHX was present in addition to LatB (SI Ap- tially expressed in response to LatB, we searched for cis-regulatory pendix, Fig. S1B), suggesting that new protein synthesis is re- elements that might control their transcription. First, using the quired for at least some aspects of the LatB response. MEME program (SI Appendix, Materials and Methods), we searched Because it seemed likely that the transcriptional response to for DNA motifs that are significantly enriched in the regions up- LatB would involve multiple genes encoding proteins of the actin stream of the annotated transcription start sites (“TSS-upstream cytoskeleton, we extracted the data for 18 such genes from the regions”). Although searches using the sets of genes up-regulated experiment of Fig. 1A. In addition to NAP1 and IDA5 (as reported in the ida5-1, lat1, lat2,orlat3 mutants did not return any significant previously; ref. 20), genes encoding cofilin, profilin, and a formin hits, a DNA motif with a core conserved sequence (Fig. 2A and SI were significantly up-regulated in wild-type cells (by ∼20-fold, ∼7- Appendix,Fig.S3A)wasfoundtobeenrichedinthe250-bpTSS- fold, and ∼4-fold, respectively) but not in the ida5-1 mutant (Fig. upstream regions of the genes up-regulated in wild type; a very 1B); the possible significance of these results is considered further similar sequence was found to be enriched in the genes up-

below. The other 13 genes showed no significant up-regulation or regulatedinanap1 mutant (SI Appendix, Materials and Methods). CELL BIOLOGY even a mild down-regulation. We also examined a set of 21 genes We designated this motif the “LAT-response element” (LRE). We encoding proteins of the microtubule cytoskeleton; they also next used the FIMO algorithm to search for LRE sequences in showed little or no specific response to LatB (Dataset S1). various gene sets and genomic regions. As expected, this analysis revealed an enrichment of LREs in the TSS-upstream regions of Role of the LAT Pathway and of a NAP1-Mediated Negative-Feedback genes up-regulated by LatB (Fig. 2B,rows1–4), and this enrichment Loop in LatB-Regulated Gene Expression. The induction of NAP1 was abolished by randomization of the DNA sequences (Fig. 2B, (but not of IDA5) by LatB requires the LAT1, LAT2, and LAT3 rows 7–9). A similar enrichment was observed using expression data gene products (the “LAT pathway”; ref. 20). To ask if the LAT from the nap1 mutant, but not with the data from the lat1-lat3 or pathway is involved in the broader transcriptional response to ida5 mutants (SI Appendix,TableS1),andnoenrichmentofLREs LatB, we carried out an RNA-seq analysis using two clones of was detected in LatB-down-regulated genes (Fig. 2B,rows5and6). each relevant genotype and a 90-min exposure to LatB (Materials The strongest positional enrichment of LREs was found im- and Methods, RNA-seq Experiment 3). We then called differentially mediately upstream of the annotated TSSs (SI Appendix, Table expressed genes by treating the pairs of clones as biological repli- S1). Interestingly, across all genes, LRE sequences are un- cates. With a false discovery rate (FDR)-adjusted P value derrepresented in the regions immediately upstream of the TSSs of <0.05 and a fold change of ≥2 as cutoffs, 801 genes were called as compared with random expectation, increasing the significance up-regulated (Fig. 1C) and 583 genes as down-regulated during the of the enrichment observed upstream of the LatB-induced genes. LatB treatment of wild-type cells. With a more stringent fold- Like the previously reported zygotic-response element (31), the change cutoff of ≥4, there were 222 genes up-regulated and LREs are also enriched in the 5′-UTRs and first introns of LatB- 35 down-regulated. We then examined the expression patterns of responsive genes (SI Appendix, Table S1), but no such enrich- these genes in the mutants. Of the 222 genes up-regulated ≥fourfold, ment was detected in the coding sequences, introns other than none was called as differentially expressed in the ida5-1 mutant; like the first, 3′-UTRs, or downstream (presumed terminator) re- NAP1, most of these genes already showed modestly elevated ex- gions. Although these observations may in part reflect mis- pression in ida5-1 cells in the absence of LatB treatment (Fig. 1D). annotation of the TSSs, we confirmed by examination of the In the lat mutants, only 28% (lat3)to46%(lat1) of the 222 genes available expressed-sequence-tag sequences that in many cases were called as differentially up-regulated, and even in many of these the LREs are indeed within the transcribed regions. Thus, be- cases, the absolute levels of expression were diminished (Fig. 1 D cause the method used to discover the LRE motif focused on the and E). (For example, the distribution of expression levels in the lat3 TSS-upstream regions, the observation of enrichment also in the mutant differed significantly from that in wild type: Kolmogorov– 5′-UTRs and first introns suggests that the motif can activate Smirnov test D = 0.70, P = 9.2E-48.) In contrast, in the nap1 mu- transcription from an upstream TSS. tants, all of the 222 up-regulated genes were called as differentially To ask if the LRE plays a functional role in LatB-induced gene expressed, and, in most cases, their expression during LatB treatment expression, we first analyzed the levels of such expression as a was significantly enhanced relative to wild type (Fig. 1 D and E; function of the numbers of LREs in the TSS-upstream regions Kolmogorov–Smirnov test D = 0.55, P = 7.6E-30), suggesting the and 5′-UTRs. A genome-wide search identified 5,046 genes with presence of an NAP1-dependent negative-feedback mechanism. at least one LRE, and we observed a positive correlation be- To explore this possibility further, we conducted a similar tween the number of LREs and the level of LatB-induced ex- RNA-seq experiment but over a longer time course. In wild-type pression in wild-type cells (Fig. 2C, WT). The correlation was cells, all of the 222 genes up-regulated ≥fourfold showed sharp also present in nap1 mutants but was absent in lat3 mutants (Fig. declines in transcript levels at time points beyond 90 min (SI 2C), suggesting a specific connection between the LAT pathway Appendix,Fig.S2A). In striking contrast, transcript levels for and the LREs. When the expression patterns of the 43 genes nearly all of these genes remained high even after 300 min of LatB with five or more upstream LREs were examined in different treatment in the nap1 mutants (SI Appendix,Fig.S2A), supporting mutants, many of them showed patterns very similar to that of the hypothesis of a NAP1-dependent negative-feedback loop. NAP1: modest and constitutive up-regulation in ida5, diminished

Onishi et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 24, 2021 A 2 C Number of LREs in TSS-upstream region/5’-UTR vs. Expression 0.8 1 WT bits All 16332 genes ≥ 1 LREs: 5046 genes; P = 2.6E−09 0.6 ≥ 2 LREs: 1564 genes; P = 1.1E−13 0 ≥ 3 LREs: 457 genes; P = 1.3E−14 ≥ 4 LREs: 136 genes; P = 2.8E−13 ≥ 5 LREs: 43 genes; P = 1.2E−09 B 0.4 ≥ 6 LREs: 17 genes; P = 1.4E−07 ≥ 7 LREs: 5 genes; P = 0.0030 Frequencies of LREs in 250-bp TSS-upstream regions 0.2 Gene sets Number LRE LRE P -value of genes counts /kb 1 All genes 17,732 4621 1.04 0 0.6 nap1 2 ≥8 Up 78 120 6.156.5 E-52 Density lat3 P P 3 ≥4 Up 222 223 4.02 1.5 E-62 = 6.3E−10 = 0.18 P = 2.3E−19 P = 0.14 ≥ 482 Up 01 445 2.22 1.0 E-48 0.4 P = 3.2E−18 P = 0.49 5N≥2 Down 583 122 0.84 .S.P = 1.7E−14 P = 0.068 P P ≥ = 7.6E−09 = 0.71 6N4 Down 35 6 0.69 .S.P = 1.8E−07 P = 0.69 7N≥8 Up, randomized 78 22 1.13 .S.0.2 P = 0.0031 P = 0.64 8N≥4 Up, randomized 222 51 0.92 .S. 9 ≥2 Up, randomized 801 202 1.01 N.S. 0 −4 −2 0 2 4 6 8 −4 −2 0 2 4 6 Log fold-change -/+ LatB 2 DEF Venus-based reporter assay Complementation of nap1-1 by PNAP1 PNAP1WT:NAP1 or PNAP1∆LRE1:NAP1 NotI LRE2 LRE1 NAP1 No LatB 1.0 μM LatB 5’ UTR CDS -543 -191 +1 ∆LRE1 PNAP1 PNAP1 P :NAP1 NotI LRE2 NAP1 K-S test value 5’ UTR CDS 0.00024 0.0092 0.029

∆LRE1 PNAP1 PVIPP2 SpeI :NAP1 NotI VIPP2 5’ UTR VENUS-3FLAG

2xLRE LREs fluorescence Venus (A.U.) in Increase PVIPP2 SpeI 21 Control NotI VIPP2 0 1000 2000 3000 4000 5000 5’ UTR VENUS-3FLAG

No-Venus PNAP1 PNAP1 PVIPP2 PVIPP2 2xLRE control :VENUS-3FLAG

Fig. 2. Identification and functional analysis of a cis-regulatory element for LAT-pathway-responsive gene expression. (A) Logo representation of the LRE consensus sequence that was identified by MEME in the TSS-upstream regions of genes highly induced by LatB in wild-type cells (SI Appendix, Materials and Methods). (B) Enrichment of LREs in TSS-upstream regions of genes up-regulated by LatB; probabilities of the observed enrichment’s occurring by chance given the background expectations (Fisher’s exact tests) are indicated. N.S., not significant. Row 1, background LRE discovery rate in the TSS-upstream regions

of all genes. Rows 2–4, enrichment of LREs in TSS-upstream regions of genes induced to varying extents (PADJ < 0.05) by LatB. Rows 5–6, lack of enrichment of LREs in TSS-upstream regions of genes repressed by LatB to varying extents. Rows 7–9, lack of enrichment of LREs when the sequences used in rows 2–4 were randomized. (C) Correlation between numbers of LREs and levels of transcriptional induction by LatB in wild-type and nap1 but not in lat3 strains. Smoothened density histograms of genes with the indicated numbers of LREs in the upstream regions (250-bp upstream of the annotated TSSs plus 5′-UTRs)

were plotted as functions of the log2 fold changes in expression in response to LatB addition (0 versus 90 min). P values indicate the probabilities (from Kolmogorov–Smirnov tests) that a given distribution is equivalent to that for all genes. (D–F) Evidence that the NAP1 LREs can be both sufficient and nec- essary for transcription in response to LatB. (D) Schematic representations of the original and modified upstream regions of NAP1 and VIPP2 in the reporter

constructs used (see SI Appendix, Materials and Methods and Fig. S3C for details). PNAP1 (pTJ001 and pTJ008): Six LREs found by FIMO (P < 0.0001) in the NAP1 2xLRE ΔLRE1 upstream region are indicated by arrows; LRE1 and LRE2 were used to construct PVIPP2 . PNAP1 (pTJ002 and pTJ009): A 54-bp deletion removes five of 2xLRE the six LREs in the NAP1 upstream region. PVIPP2 (pRAM103): The VIPP2 (Cre11.g468050) upstream region lacking LREs. PVIPP2 (pMO606): The VIPP2 upstream region in which two LREs from NAP1 have been inserted immediately upstream of a putative TATA box. (E) Venus-3FLAG expression from reporter constructs with various TSS-upstream-regions sequences. For each construct, 48 randomly selected transformants were grown in liquid Tris-acetate-phosphate (TAP), and Venus fluorescence was measured using a plate reader before and after a 6-h treatment with 10 μM LatB (strong lot). Values above 1,000 arbitrary units (A.U.) are highlighted in red. Where indicated, the significance of the difference in distributions was inferred using the Kolmogorov–Smirnov test. Constructs used: pMO448 (which expresses only the selection marker), pTJ001, pTJ002, pRAM103, and pMO606. (F) Relative abilities of the normal and LRE- deficient NAP1 upstream regions to drive NAP1 expression as judged by rescue of the LatB sensitivity of a nap1 mutant. The mutant strain was transformed with the indicated constructs (from plasmids pTJ008, pTJ009, and pRAM103), and 48 randomly chosen clones for each construct were spotted on TAP plates with and without 1.0 μM LatB (strong lot).

or complete loss of response in lat1-lat3, and enhanced expression promotion of transcription in response to LatB. First, we cloned in nap1 (SI Appendix,Fig.S3B). Taken together, these results sug- the 543-bp TSS-upstream region (which contains six LREs) and gest that the LREs play a role in gene induction by LatB treatment placed it upstream of a codon-optimized VENUS-3FLAG se- in a LAT-pathway-dependent manner. quence (Fig. 2D, PNAP1 and SI Appendix, Fig. S3C). Many We next used two assays to ask more directly if the LREs in transformants containing this construct showed clear induction the region upstream of NAP1 are indeed important for the of Venus-3FLAG in response to LatB treatment, as judged by a

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1721935115 Onishi et al. Downloaded by guest on September 24, 2021 fluorescence plate-reader assay of 48 clones (Fig. 2E), Western wild-type cells (Fig. 3 B, 1) despite the induction of IDA5 tran- blotting (SI Appendix, Fig. S3D), and fluorescence microscopy scription (Fig. 1B and ref. 20) and the accumulation of NAP1 (SI Appendix, Fig. S3E). [Note that the expression of transgenes protein (Fig. 3 B, 4) that occur at the same time. This degra- from two-promoter constructs like the ones used here is highly dation was substantially more rapid than that occurring by variable among transformants in Chlamydomonas (32).] How- baseline protein turnover when new protein synthesis was ever, when we deleted a 54-bp region immediately upstream of blocked by CHX (Fig. 3 B, 2). Furthermore, it was slowed when ΔLRE the TSS that contains five of the six LREs (Fig. 2D, PNAP1 ), CHX was added together with LatB (Fig. 3 B, 3), suggesting that the transformants showed little or no induction of Venus- new protein synthesis is important for the degradation pathway. 3FLAG in response to LatB treatment (Fig. 2E and SI Appen- We hypothesized that the newly synthesized proteins included dix, Fig. S3 D and E), indicating that the LRE cluster is impor- the products of the specifically up-regulated ubiquitin/proteasome- tant for the LatB response. Second, when we used the same two pathway genes. This hypothesis was supported by three additional promoters to drive expression of NAP1 itself, most transformants lines of evidence. First, IDA5 degradation was largely eliminated containing the full-length PNAP1:NAP1 showed rescue of a nap1-1 in the lat mutants, which do not up-regulate the ubiquitin/pro- ΔLRE1 mutant, whereas most transformants containing PNAP1 :NAP1 teasome components, but not in a nap1 mutant, which does (Fig. (lacking the 54-bp region) did not (Fig. 2F). 3C). Second, IDA5 degradation was greatly reduced when a strain Finally, to test further whether LREs can be sufficient to drive carrying a temperature-sensitive mutation in a gene for an es- LatB-responsive expression, we used a PVIPP2:VENUS-3FLAG sential proteasome subunit was exposed to LatB at its restrictive construct that contains no endogenous LREs (Fig. 2D, PVIPP2) temperature (Fig. 3D). Third, a dose-dependent reduction in (33) and showed little induction of Venus-3FLAG above back- IDA5 degradation was seen when wild-type cells were exposed ground when transformants were exposed to LatB (Fig. 2E and SI to LatB in the presence of the proteasome inhibitor MG-132 Appendix,Fig.S3F). When two LREs from the NAP1 upstream (Fig. 3E). Importantly, MG-132 treatment did not block LatB region were inserted immediately upstream of the TSS in this induction of NAP1 (Fig. 3E), indicating that the inhibitor did not 2xLRE construct (Fig. 2D, PVIPP2 ), transformants showed LatB- simply abrogate all response to LatB. responsive Venus expression (Fig. 2E and SI Appendix,Fig.S3F). Ubiquitination almost invariably occurs on lysine residues. By Taken together, the results suggest that LREs can be both sequence alignment, we found eight lysines in IDA5 that are not sufficient and necessary for LatB-induced gene expression, at present in NAP1 (Fig. 4A). To ask if these residues might ac- least in appropriate sequence contexts, and the global survey count for the fact that IDA5 is degraded during exposure to LatB suggests that such LRE-mediated control may account for a sub- while NAP1 is not, we replaced these lysines in IDA5 with the

stantial fraction of the overall transcriptional response to LatB. corresponding residues from NAP1. Tetrad analysis showed that CELL BIOLOGY the IDA58Kmut transgene rescued the inviability of ida5-1; nap1-1 Induction by LatB of Genes Encoding Proteins of the Ubiquitin- double mutants (Fig. 4B), indicating that the IDA58Kmut protein Proteasome System. To explore the functional significance of retains essential actin functions. However, upon LatB treatment, the widespread gene induction by the LAT pathway, we per- this protein was stable, unlike either endogenous or transgenic formed a (GO)-term analysis on the 363 “LAT- wild-type IDA5 (Fig. 4C, lanes 2, 4, and 6), suggesting that target” genes that were up-regulated ≥twofold in wild type but ubiquitination of one or more of the eight lysines targets the not in lat3 mutants (Fig. 1C). Because of the paucity of anno- protein for degradation, although we have not yet been able to tations in the Chlamydomonas database, we relied primarily on demonstrate such ubiquitination biochemically because of the lack annotations in the yeast Saccharomyces cerevisiae to calculate of a reliable method to purify IDA5. Interestingly, the lack of enrichment of GO terms in the up-regulated genes. To do so, we degradation of IDA58Kmut protein did not prevent the up-regulation identified S. cerevisiae homologs of the Chlamydomonas gene of NAP1 during exposure to LatB (Fig. 4C, lane 6), suggesting that products by using a method we named R/UBH (reciprocal or IDA5 degradation is not a prerequisite for function of the LAT unidirectional best blast hit), which looks for pairs of genes in pathway of transcriptional induction. In addition, IDA58Kmut,but two organisms whose products form a best BLAST hit in at least not wild-type IDA5 expressed from the same promoter, conferred one direction and a statistically meaningful BLAST hit (E < 0.1) partial resistance to LatB in in ida5-1; nap1-1 cells (Fig. 4D), pos- in the other direction (SI Appendix, Materials and Methods). sibly because the undegraded IDA5 helps to drive the G-actin/ Despite the large evolutionary distance, we found in this way F-actin equilibrium toward F-actin in the presence of low concen- 2,651 candidate S. cerevisiae homologs of 6,473 Chlamydomonas trations of LatB. proteins (some yeast proteins had more than one Chlamydo- monas homolog), including 148 of the 363 LAT-target gene Roles of Profilin and Cofilin in Modulating the Degradation of IDA5 products. The higher fraction of yeast homologs among the Actin. Profilin mRNA (Fig. 1B, PRF1) and protein (Fig. 5A) were LAT-target gene products (39%) than among all proteins (15%) modestly induced when wild-type cells were treated with LatB. indicates a significant enrichment of highly conserved genes To explore the possible role(s) of profilin in actin homeostasis among the LAT targets (χ2 test P < 0.0001). The GO-term- during exposure to LatB, we used the prf1-1 temperature- enrichment analysis of the 148 proteins found a highly signifi- sensitive-lethal mutant (ref. 34, there called “div68-1”). In- cant enrichment of terms related to protein degradation terestingly, although prf1-1 cells are viable at 21 °C and inviable mediated by the ubiquitin-proteasome system and essentially at 33 °C, they had a barely detectable level of profilin protein nothing else (Fig. 3A and SI Appendix,TableS2). Independent even at the lower temperature (Fig. 5B), indicating that the searches against the Chlamydomonas, Arabidopsis thaliana,and mutation destabilizes the protein at both temperatures even mouse databases (SI Appendix, Materials and Methods)gave though the effect is lethal only at the higher temperature. Sur- similar results, indicating that nothing of consequence had been prisingly, IDA5 was essentially absent in prf1-1 cells even when missed because of an absence of related genes in yeast. Most of grown at 21 °C in the absence of LatB, while NAP1 was highly these genes were not induced in the pH-shock and CHX- elevated (Fig. 5C), suggesting that IDA5 degradation can be treatment gene sets (SI Appendix,Fig.S1), suggesting that the triggered by a lack of binding to profilin and that the lack of induction is a specific response to LatB. IDA5, and thus also of F-IDA5, can promote NAP1 up- regulation even in the absence of drugs. Ubiquitin/Proteasome-Dependent Degradation of IDA5 Actin During Because ida5-1; nap1-1 double mutants (lacking both actins) the Response to LatB. The general up-regulation of genes of the are inviable (20), we expected that prf1-1; nap1-1 double mutants ubiquitin/proteasome pathway during exposure of cells to LatB would also be inviable for the same reason, and this was indeed suggests that proteolysis of one or more proteins may play an the case (Fig. 5D, rows 1 and 2 and E, 1). Because the LAT important role in actin homeostasis under these conditions. pathway is essential for the induction of NAP1 synthesis, we also Indeed, IDA5 was rapidly degraded during LatB treatment of expected that prf1-1 lat double mutants would lack both actins

Onishi et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 24, 2021 and thus be inviable, but, surprisingly, this was not the case (Fig. A 35 LAT-target genes whose yeast homologs are 5D, rows 3–5 and E, 2). The apparent discrepancy might be annotated with GO:Proteolysis explained if the lat mutations rescue IDA5 levels in prf1-1 cells lat3 nap1 WT Yeast homolog by because they block up-regulation not only of NAP1 but also of LatB: –+–+–+ R/UBH and annotation the ubiquitin-mediated-proteolysis genes (discussed above). Di- * * Cre06.g275650 RPN3 Proteasome lid * * Cre06.g278256 RPN8 Proteasome lid rect examination of IDA5 and NAP1 levels in the single- and * * Cre10.g424400 PUP1 Proteasome core * * Cre09.g396400 UBI4 Ubiquitin double-mutant strains supported this model: Whereas prf1-1 * * Cre04.g216600 RPT6 Proteasome base single-mutant cells had high levels of NAP1 and undetectable * * Cre09.g402450 RPT3 Proteasome base * * Cre06.g279000 PRE3 Proteasome core IDA5, prf1-1 lat double-mutant cells had the opposite pattern * * Cre08.g373250 PRE8 Proteasome core * * Cre17.g727950 RPN2 Proteasome base (Fig. 5F). Taken together, the results suggest that the LAT * * Cre16.g676197 RPN1 Proteasome base * * Cre11.g475400 RPN11 Proteasome lid pathway targets IDA5 for destruction even in the absence of * * Cre08.g366400 RAD23 Ub-like N terminus LatB (probably through induction of the ubiquitin/proteasome * * Cre14.g625400 RPT1 Proteasome base * * Cre16.g693700 UBC4 E2 Ub-conjugating protein system), and that profilin has a previously unknown role in * * Cre11.g478240 RPN5 Proteasome lid * * Cre04.g212401 RAD6 E2 Ub-conjugating protein protecting G-IDA5 from such degradation. * * Cre01.g030800 RPN6 Proteasome lid * * Cre01.g019850 AFG3 AAA family ATPase Another gene that was highly induced by LatB treatment was * * Cre01.g012450 RSP5 NEDD4 family E3 Ub ligase COF1, encoding the F-actin-severing protein cofilin (Fig. 1B and * * Cre07.g324400 VPS24 ESCRT-III subunit * * Cre02.g095093 SEM1 Proteasome rlid SI Appendix, Fig. S4). We described previously a single recessive * * Cre07.g336200 UBP15 Ub-specific protease * * Cre12.g543450 HRT1 SCF Ub-ligase subunit suppressor of the LatB sensitivity of a nap1 null mutation (20), * * Cre17.g734400 CDC53 Cullin and we have now shown that this mutation causes a Leu-to-Pro * * Cre06.g281350 PIM1 ATP-dependent Lon protease * * Cre12.g501200 SKP1 SCF Ub-ligasesubunit substitution at position 18 in COF1 (Materials and Methods and * * Cre01.g030550 UBP14 Ub-specific protease * * Cre06.g278116 POF1 NMNase SI Appendix, Fig. S4). Suppression of nap1 LatB sensitivity by * * Cre06.g304000 RAD16 DNA repair protein * * Cre14.g614400 RAD16 DNA repair protein cof1-1 might arise if the loss of F-IDA5 and consequent degra- * * Cre13.g580600 ATE1 Arginyltransferase dation of IDA5 were slowed by reduced severing of F-IDA5 * * Cre10.g443950 UFD2 Ub-Ub ligase * * Cre12.g517850 SSM4 E3 Ub-protein ligase filaments. Consistent with this hypothesis, IDA5 was substan- * * Cre16.g676350 CDC48 AAA family ATPase * **Cre15.g640101 GRR1 SCF Ub-ligase subunit tially stabilized in a LatB-treated cof1-1 strain (Fig. 5G). In wild- Log fold-change from WT at t=0: type cells, the induction of COF1 by LatB might speed F-IDA5 2 B −10 −5 0 5 10 disassembly, and thus IDA5 degradation, thereby promoting the replacement of F-IDA5 by F-NAP1. Time WB: IDA5 WB: NAP1 CBB (min): 03015 60 120 0 301560 120 0 301560 120 1 4 Role of IDA5 Degradation in Actin Homeostasis During Exposure to LatB LatB. To investigate the possible importance of IDA5 degradation in 2 5 the response to actin depolymerization by LatB, we used strains CHX carrying a nap1 null mutation and expressing wild-type NAP1 from the constitutive TUB2 promoter, an arrangement that simplifies the LatB 3 6 CHX analysis by removing transcriptional induction of NAP1 as a factor. In the absence of other mutations, such a strain showed a partial WT lat1-1 lat2-1 lat3-1 nap1-2 resistance to LatB that was distinctly less than that of wild type (Fig. C LatB: –+ –+ –+ –+ –+ 6A, columns 1–3 and ref. 20), probably because the levels of WB: IDA5 NAP1 were less than the induced level in wild-type cells exposed to LatB (Fig. 6B; compare lanes 5, 7, and 8 to lanes 2 and 6). When WB: IDA5 LatB (10 μM): – ++++ thestrainalsoharboredalat3 mutation, its LatB resistance was DE21°C 33°C MG132 (μM): 0 0 10 30 100 further decreased (Fig. 6A, column 4 and ref. 20). This observation LatB: –+–+ cannot be explained by a failure of NAP1 induction as a result of the WB: IDA5 lat3 mutation, because NAP1 in this strain is only expressed ec- WT topically from the TUB2 promoter; therefore, we hypothesized that WB: NAP1 rpt5ts it might be due to failure of expression of other LatB-induced CBB genes. Specifically, the decreased resistance might be due to re- duced expression in the lat3 mutant of the ubiquitin-proteasome system, leading to increased persistence of LatB-bound IDA5 Fig. 3. Degradation of IDA5 through a mechanism dependent on the LAT and ubiquitin/proteasome pathways. (A) LAT-pathway-dependent induction of genes monomers. Indeed, elimination of IDA5 from these strains by an whose S. cerevisiae homologs are annotated with the GO term “Proteolysis.” Data ida5-1 mutation resulted in greatly enhanced LatB resistance, are from RNA-seq Experiment 3 (cf. Fig. 1 C–E). (B) Rapid degradation of whether or not LAT3 was present (Fig. 6A, columns 5 and 6). Taken IDA5 during exposure of wild-type cells (strain CC-124) to LatB. Levels of together, these results suggest that proteolytic elimination of LatB- IDA5 and NAP1 were analyzed by Western blotting using an anti-actin bound IDA5 monomers can facilitate actin homeostasis, probably by antibody (SI Appendix, Materials and Methods) and then (after stripping reducing interference with the assembly and/or function of F-NAP1. the blots) an anti-NAP1 antibody after treatment of cells for various times with 10 μM LatB (strong lot), 10 μg/mL CHX, or both. For each sample, 30 μgof Discussion whole-cell extract were analyzed; Coomassie Brilliant Blue (CBB) staining of the A Broad Transcriptional Response to Disturbance of F-Actin. De- membranes provides a loading control. Blots are numbered for ease of refer- polymerization of F-IDA5 by LatB leads to strong transcriptional ence in the text. (C) Dependence of IDA5 degradation on the LAT pathway. induction of the gene encoding the divergent actin NAP1 (20) Wild-type (CC-124) and mutant strains of the indicated genotypes were grown μ and of several hundred other genes (this study). In most cases, at 25 °C and treated with 10 M LatB (strong lot) for 2 h. Samples were ana- this induction depends on the LAT pathway involving the LAT1, lyzed by Western blotting as in B.(D and E) Dependence of IDA5 degradation LAT2, and LAT3 gene products and appears to be under the on the ubiquitin/proteasome pathway. Samples were analyzed by Western blotting as in B.(D) Lack of IDA5 degradation in a mutant with a temperature- control of a negative-feedback loop that requires NAP1 (and sensitive proteasome subunit. Wild-type (CC-124) and rpt5-1 strains were thus also the LAT pathway). Several possible models might ex- plain these observations. First, the pathway might be activated by grown at 21 °C, each culture was split into two, and the subcultures were in- – cubated at 21 °C or 33 °C for 6 h before treatment with 10 μM LatB (weak lot) the transient overabundance of G-IDA5 (or G-IDA5 LatB for 2 h. (E) Dose-dependent inhibition of IDA5 degradation by a proteasome complexes). However, this model is inconsistent with the up- inhibitor. Wild-type (CC-124) cells were grown at 25 °C and treated (or not) regulation of NAP1 in the ida5-1 and prf1-1 mutants, which with 10 μM LatB (strong lot) and with the indicated concentrations of the lack G-IDA5. Second, the pathway might be activated by proteasome inhibitor MG-132 for 2 h. the subsequent loss of IDA5 protein. However, this model is

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1721935115 Onishi et al. Downloaded by guest on September 24, 2021 A B

CD CELL BIOLOGY

Fig. 4. Importance of IDA5-specific lysines for its degradation during LatB exposure. (A) Absence in NAP1 of eight lysines (marked by #) that are conserved in IDA5 and human skeletal α-actin; a single lysine is found specifically in NAP1 (marked by *). (B) Provision of essential actin function(s) by IDA58Kmut, which lacks 8Kmut the eight lysines that are absent in NAP1. An mt+ ida5-1 strain was transformed with PTUB2:IDA5 (from pMO589) or PTUB2:IDA5 (from pMO600). After confirmation of transgene expression by Western blotting, a transformant of each genotype was crossed with a mt− nap1-1 strain, tetrads (shown in columns) were dissected, and segregants were genotyped by allele-specific PCR (for ida5-1), sensitivity to 10 μM LatB (strong lot; for nap1-1), and paromomycin re- 8Kmut sistance (for the transgenes). Arrowheads, ida5-1; nap1-1; PTUB2:IDA5 and ida5-1; nap1-1; PTUB2:IDA5 segregants. All inviable segregants were inferred to be ida5-1; nap1-1 based on the presumed 2:2 segregation of all markers in the tetrads. (C) Lack of IDA58Kmut degradation, but up-regulation of NAP1, during exposure of cells to LatB. Strains of the indicated genotypes were incubated with or without 3.0 μM LatB (strong lot) for 2 h at 25 °C, and protein levels were analyzed as in Fig. 3B. Results for IDA5, IDA58Kmut, and NAP1 were similar for each of two transgenic lines of each genotype. (D) Partial resistance to LatB of nap1-1 cells expressing IDA58Kmut. Segregants of the indicated genotypes (taken from the crosses shown in B) were spotted in fivefold dilution series on TAP plates without LatB or with LatB (strong lot) at the indicated concentrations and grown at 25 °C for 4 d.

inconsistent with the evidence that NAP1 is induced when with small numbers of LREs do not respond strongly to LatB, IDA5 degradation does not occur (the IDA58Kmut mutant and genes with multiple, closely spaced LREs are likely to do so, wild-type cells treated with the proteasome inhibitor MG132). perhaps because the actual mechanism involves some form of Thus, the most attractive model (Fig. 7) appears to be that the cooperativity of transcription-factor binding. Such homotypic site structure and/or function of F-actin (either F-IDA5 or F-NAP1) clustering is a commonly observed feature of transcriptional suppresses the transcriptional-response pathway. Upon LatB regulation in diverse systems. We have not yet identified a can- treatment of wild-type cells, F-IDA5 is lost, which activates the didate transcription factor for binding to this motif, and the pathway; as NAP1 is produced, F-NAP1 assembles, shutting off amino acid sequences of LAT1, LAT2, and LAT3 do not suggest the pathway. This model also appears consistent with the ob- that any of them has the ability to bind specific DNA motifs. servations on the ida5-1 and prf1-1 mutants. In the former, it There have been few, if any, genome-wide analyses of tran- appears that a low level of constitutive pathway activation is suf- scriptional responses to perturbations of F-actin. However, it ficient to maintain levels of NAP1 adequate for cell function, and has been reported that in mammalian cells, actin and actin- because F-NAP1 does not depolymerize upon LatB addition, binding proteins participate directly in the transcriptional reg- there is no additional transcriptional response upon exposure to ulation of many genes in response to shocks to the actin the drug. In the prf1-1 mutant, the instability of IDA5 leads to a cytoskeleton (35–38). In addition, latrunculin treatment has been loss of F-IDA5, and thus activation of the pathway, until sufficient shown to activate MAP kinases in yeasts (27, 29). Although most F-NAP1 is present to achieve a steady state. characterization of the downstream responses to activation of the Examination of the sequences upstream of the LatB-induced yeast kinases has focused on nontranscriptional regulation, an ad- genes revealed a potential cis-acting regulatory site, the LRE ditional transcriptional component may well exist. In land plants, motif, variants of which were found in the promoters, 5′-UTRs, pathogen infection can cause disturbances in F-actin structures, and introns of many such genes. We found that these motifs are which in turn induce genes related to the innate-immune re- highly enriched near the TSSs and that there was a strong cor- sponse (39, 40). Thus, it appears that the ability to recognize relation between the number of LREs and the induction of ex- and respond to perturbations of the actin cytoskeleton arose pression by LatB. Thus, although the majority of the 5,046 genes early in eukaryotic evolution.

Onishi et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 24, 2021 ABCDWT prf1-1 Growth at 21°C Tetrads Live:dead Growth at: 21° 33° 21° 33° Cross scored 4:0 3:1 2:2 prf1-1 Time in Profilin in WT WT 11prf1-1 x WT 20 901 LatB (min): 0 3 10 30 60 90 120 Short 2 prf1-1 x nap1-1 26 8 10 8 WB: WB: IDA5 3 prf1-1 x lat1-5 36 36 0 0 WB: PRF1 PRF1 Long WB: NAP1 4 prf1-1 x lat2-1 21 18 3 0 5 prf1-1 x lat3-1 24 20 3 1

EFprf1-1 (no IDA5) prf1-1 (no IDA5) G x x nap1-2 (no NAP1) lat3-1 (no NAP1) WT cof1-1 WT lat1-5 prf1-1 prf1-1 lat1-5WT lat2-1 prf1-1 prf1-1 lat2-1WT lat3-1 prf1-1 prf1-1; lat3-1 LatB: –+ –+ 12WB: IDA5 WB: IDA5

WB: NAP1 WB: NAP1

Fig. 5. Roles of profilin and cofilin in preventing and accelerating the degradation of IDA5. (A and B) Analysis of profilin levels by Western blotting. For each sample, 15 μg of whole-cell extract were subjected to Western blotting using an antibody to Chlamydomonas profilin. (A) Induction of profilin expression by LatB. Wild-type strain CC-124 was treated with 10 μM LatB (weak lot) at 25 °C for the indicated times. (B) Nearly total absence of profilin in a temperature- sensitive profilin mutant even when grown at a temperature permissive for growth. Wild-type (CC-124) and prf1-1 strains were grown at 21 °C or shifted to 33 °C for 6 h. Images of the same blot with short and long exposure times are shown. (C) Loss of IDA5 and induction of NAP1 in the temperature-sensitive profilin mutant even when grown at 21 °C. (D and E) Synthetic lethality of the profilin mutation with nap1-1 but not with lat1, lat2,orlat3 mutations. (D)For each of the indicated crosses, tetrads were dissected and segregants scored for viability. (E) Representative tetratype tetrads from the indicated crosses were imaged after 2 d at 21 °C. Genotypes of the segregants were determined based on temperature sensitivity (prf1-1) and LatB sensitivity (nap1-1 and lat3-1), and the double-mutant segregants are indicated by the arrowheads. The prf1-1; nap1-1 cells (plate 1) divided several times after germination (presumably due to maternal mRNA and/or protein) before lysing. (F) Rescue of IDA5 protein levels in the profilin mutant by lat mutations. A tetratype tetrad from each relevant cross (D, rows 3–5) was analyzed by Western blotting as in Fig. 3B.(G) Reduced degradation of IDA5 during LatB treatment of a cofilin mutant. Strains of the indicated genotypes were grown at 25 °C with or without treatment for 2 h with 10 μM LatB (weak lot).

Regulation of Actin Levels by Ubiquitin-Proteasome-Mediated Proteolysis and F-IDA5, might itself constitute enough of a stress to the F-actin and Protection from Proteolysis by Profilin. In GO analyses of the cytoskeleton to trigger activation of the LAT pathway, resulting in genes induced by LatB through the LAT pathway, the only highly up-regulation of the ubiquitin-proteasome system and enhancement enriched terms were related to theubiquitin-proteasomesystem. of IDA5 degradation. In this regard, it is of interest that our pre- Many genes encoding components of E3-ubiquitin ligases (including liminary RNA-seq analysis of a prf1-1 mutant showed gene- multiple proteins of the SCF complex) and the proteasome itself expression changes with strong similarity to those seen in wild- were up-regulated 2- to 10-fold. Such levels of up-regulation seem type cells treated with LatB. To our knowledge, such a role of very likely to be significant given that the expression of proteasome profilin in protecting actin from degradation has not been reported components is frequently tightly regulated by negative-feedback in other systems. mechanisms that keep their overall levels constant, so that changes We hypothesize that IDA5 degradation in response to LatB of this magnitude are rarely observed (41). eliminates nonpolymerizable G-IDA5–LatB complexes that IDA5 is a likely target of the up-regulated ubiquitin-proteasome could competitively interfere with NAP1 polymerization. system. In LatB-treated cells, IDA5 is rapidly degraded in a pro- This hypothesis is supported by our observations on a nap1 cess that depends on proteasome function. IDA5 degradation PTUB2:NAP1 strain, which has lower levels of NAP1 than a wild- under these conditions also depends on the LAT pathway type strain whose endogenous NAP1 gene has been induced by and new protein synthesis, presumably because components of LatB treatment. In the absence of other mutations, the nap1 the ubiquitin-proteasome system must be up-regulated to achieve PTUB2:NAP1 strain showed only weak resistance to LatB. This efficient degradation. Blocking IDA5 degradation by replace- resistance was further weakened when the degradation of IDA5 was ment of lysine residues significantly attenuated the LatB sensi- reduced by a lat mutation, but it was restored to wild-type levels by tivity of a nap1 strain (Fig. 4D), indicating that the ubiquitination an ida5 mutation that simply eliminates IDA5 protein. of IDA5 is functionally significant. Interactions of actin with the ubiquitin-proteasome system Even in cells not treated with LatB, IDA5 is degraded (with have also been observed in other organisms. For example, in concomitant up-regulation of NAP1) when profilin function is mammals, mass-spectrometry studies found evidence that some compromised by the prf1-1 mutation. Thus, binding to profilin of the many actin isoforms are ubiquitinated in vivo (45, 46), and may help to maintain the proper folding of G-IDA5, shield it some of the putative ubiquitination sites correspond to the ly- from the ubiquitination machinery, and/or protect it from deg- sines that are present in IDA5 but not in NAP1. In addition, a radation by promoting its assembly into filaments. Consistent recombinant actin expressed in mouse muscle cells was found to with the second possibility, at least three of the eight lysine be ubiquitinated and degraded (47), and in synapses, ubiquiti- residues in IDA5 that are possible targets for ubiquitination nation was found to regulate γ-actin levels (48) and thus add an (K120, K286, and K361) are located in or near the actin–profilin important layer of regulation of the balance of actin isoforms interfaces where this has been studied (42–44). The adaptive benefit during the establishment of neuronal connections (49, 50). of PRF1 up-regulation in response to LatB may be sequestration of the transiently overabundant G-IDA5, assistance in NAP1 polymeri- Summary: The Actin-Homeostasis System of Chlamydomonas. In re- zation, or both. Interestingly, IDA5 degradation in the prf1-1 mutant sponse to the insult to their F-IDA5–based actin cytoskeletons also depends on the LAT pathway, an observation that suggests produced by LatB, Chlamydomonas cells up-regulate production several possibilities that are not mutually exclusive. First, the LAT of (i) an alternative actin, NAP1, that is insensitive to the drug; pathway might be required for production of some factor(s) involved (ii) the ubiquitin-proteasome system, which degrades LatB- in targeting of IDA5 by the ubiquitin-proteasome system even in the IDA5 complexes, probably to prevent them from interfering with absence of a major perturbation of the actin cytoskeleton. Second, the the formation of F-NAP1; (iii) profilin, which appears to pro- loss of profilin function in the prf1-1 mutant, with resulting loss of G- tect G-IDA5 from uncontrolled degradation and may promote

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1721935115 Onishi et al. Downloaded by guest on September 24, 2021 M12P to obtain the cof1-1 single mutant; the mutation was then identified A PTUB2:NAP1 nap1-2; by bulked-segregant sequencing (34). The ida5-1 strains were derivatives of CC- 3421 (obtained from the Chlamydomonas Resource Center) that had been back- nap1-2; nap1-2; lat3-1; crossed three times with CC-124 or iso10. ida5-1 had originally been isolated (as nap1-2 nap1-2 lat3-1 ida5-1 ida5-1 WT ida5) and shown to be a null mutant by Kato-Minoura et al. (15). For RNA-seq Experiment 5 (discussed below), a nap1-2 mutant that had been transformed + with a PTUB2:NAP1 construct (20) was crossed to iso10, and two NAP1 and two 0 + NAP1 PTUB2:NAP1 segregants were used for the analyses.

Transcriptome Analysis. We performed five transcriptome analyses by RNA- 1.0 − LatB seq (Dataset S1), as follows. (i) CC-124 and an ida5-1 mt strain were grown to OD750 ∼0.3 at 21 °C, and LatB (weak lot) was added to a final (μM) concentration of 10 μM. Samples (10 mL) were collected before LatB 3.0 addition and at intervals thereafter by brief centrifugation, and the pellets were frozen immediately in liquid nitrogen and stored at −80 °C. (ii) Like Experiment 1 except that strain CC-124 was treated with 10 μM LatB (weak lot), 10 μg/mL CHX, or both. In addition, a separate culture of CC- 10 124 was shifted to 33 °C and incubated at that temperature for 6 h. (iii) M11H, P11P, ida5-1 mt−, ida5-1 mt+, lat1-1, lat1-5, lat2-1, lat2-2, lat3-1, lat3-3, nap1-1,andnap1-2 strains were treated as in Experiment 1, except that 12 3456 samples were added to tubes with crushed ice and centrifuged briefly; the B PTUB2:NAP1 pellets were then transferred to 2-mL cryogenic storage tubes and frozen at −80 °C. (iv)LikeExperiment3butwithsamplestakenoveramoreextended WT ida5-1 WT nap1-2 time course. (v) Two wild-type and two PTUB2-NAP1 strains (all segregants from

LatB: –+ –+ –+ –+ the same cross; discussed above) were grown to OD750 ∼0.1 at ∼23 °C, and LatB WB: NAP1 (strong lot) was added to a final concentration of 3.0 μM. Samples (9 mL) were collected before LatB addition and at intervals thereafter as in Experiment 3. CBB To prepare samples for RNA-seq, prechilled glass beads, 500 μL phenol- chloroform, and 500 μL RNA extraction buffer (10 mM Tris·Cl, pH 8.0, 1 mM 12345678 EDTA, 0.2 M NaCl, and 0.2% SDS) were added in this order to each frozen CELL BIOLOGY cell pellet, and the cells were disrupted by vigorous vortexing. After cen- Fig. 6. Importance of IDA5 degradation for full resistance to LatB. (A) Single trifugation for 15 min at 12,000 × g, the aqueous phase was recovered; RNA cells of the indicated genotypes were placed by micromanipulation on TAP was then precipitated with ethanol, dissolved in RNase-free water, and plates containing the indicated concentrations of LatB (strong lot), and the plates further purified using the RNeasy mini kit (Qiagen) following the manu- were incubated for 3 d at 25 °C. Images are representative of at least 10 cells for facturer’s instructions. RNA concentrations were measured using a Nano- each strain and LatB concentration. The strains used in columns 3–6 are segre- Drop (Thermo Scientific), and RNA integrity was assessed using an Agilent gants from a single cross and therefore have PTUB2:NAP1 inserted at the same chromosomal location (and thus presumably expressed at approximately the 2100 Bioanalyzer using the Plant RNA assay; all RNA-integrity numbers were μ same level); columns 1 and 2 are internal controls. (B) Expression of NAP1 protein between 7.1 and 9.9. cDNA libraries were generated from 1 g total RNA per sample using a TruSeq RNA kit (Illumina) and sequenced (50-bp single-end from the endogenous locus and the PTUB2:NAP1 transgene. Strains of the in- dicated genotypes were treated with 3.0 μM LatB (strong lot) for 120 min at reads) by Genewiz using an Illumina HiSeq 2500. Alignments of reads to the 21 °C, and extracts were analyzed by Western blotting as in Fig. 3B. reference genome and read-count calculations were performed as described previously (52). For the heat-map displays, read counts for each gene were

F-NAP1 polymerization; and (iv) cofilin, which may promote the prompt removal of IDA5 by severing the LatB-sensitive fila- F-IDA5 ments (Fig. 7). Remarkably, these responses together produce an F-actin–homeostasis system so efficient that a sudden exposure Profilin of wild-type cells to a high dose of LatB produces little or no Transcriptional interruption in their growth rate (20), while within the cells the G-IDA5 response entire complement of IDA5 has been depolymerized, degraded, Cofilin pathway and replaced with NAP1. This system seems likely to have evolved LAT1 LAT2 LatB No as a defense against compounds in the environment that damage profilin LAT3 G-NAP1 the actin cytoskeleton (20), but the same system appears to operate Profilin also in the absence of drugs, for example when IDA5 protein (and thus F-IDA5) is lost in a profilin mutant. It will be interesting to see the extent to which this system operates also during other specific IDA5 degradation insults to the actin cytoskeleton in Chlamydomonas andtolearnthe degree to which the elements of this system (or analogous func- tions) are conserved in other organisms. Fig. 7. Proposed model for the F-actin–homeostasis pathway in Chlamy- Materials and Methods domonas. In normal vegetative cells, IDA5 is the only actin expressed; it cy- Strains. C. reinhardtii wild-type strains CC-124 (mt−) and iso10 (mt+, con- cles between the monomeric (G) and filamentous (F) forms with the aid of both genic to CC-124; provided by S. Dutcher, Washington University in St. Louis, profilin (which protects it from degradation and probably delivers it to a formin St. Louis) were the parental strains. Hygromycin-resistant (M11H; mt−) and for polymerization) and cofilin (which probably promotes filament turnover). In paromomycin-resistant (P11P; mt+) derivatives were obtained by trans- LatB-treated cells, binding of G-IDA5 by LatB leads to loss of F-IDA5, which in turn formation of iso10 with aph7″ and APHVIII genes followed by several activates a transcriptional pathway that up-regulates multiple genes including the backcrosses (51). The lat1, lat2, lat3, nap1, prf1-1 (originally isolated as div68- alternative actin gene NAP1 and genes encoding proteins of the ubiquitin- 1), and rpt5-1 (originally isolated as gex36-1) mutants used in this study were proteasome system. This system then degrades the G-IDA5-LatB complexes isolated previously in the CC-124 background (20, 34) and had been back- (which could otherwise interfere with the polymerization of NAP1) but not NAP1. crossed at least twice with CC-124 or iso10 (or their drug-resistant deriva- As NAP1 also does not bind LatB, it polymerizes (probably also with the aid of tives). The cof1-1 mutation was originally isolated in a phenotypic revertant profilin, as prf1-1 is a lethal mutation at high temperature) into filaments, which of a nap1-1 strain (20), which was back-crossed several times with M11H and in turn shut down the transcriptional pathway, forming a negative-feedback loop.

Onishi et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 24, 2021 first corrected for the library size and then normalized against the value for homolog, and GO-term analysis, microscopy and plate-reader analyses, and wild type at time = 0. Western blotting are described in SI Appendix, Materials and Methods. For Experiment 2, differential-expression analysis was performed using EdgeR (53) and treating the 0-min and 10-min samples as one pair of rep- ACKNOWLEDGMENTS. We thank Frej Tulin, Takako Kato-Minoura, Ritsu licates and the 90-min and 120-min samples as a second pair of replicates Kamiya, David Kovar, Chris Staiger, Arthur Grossman, Martin Jonikas, Jim (Dataset S2). For Experiment 3, differential-expression analysis was per- Umen, Prachee Avasthi, Alex Paredez, and Ryuichi Nishihama for valuable formed using DESeq2 (54) and the two replicate samples for each genotype discussions, the provision of valuable reagents, or both. We also thank and time point examined (Dataset S3). members of our laboratories for many valuable discussions and the Smoothened density histograms were generated in R using the density Chlamydomonas Resource Center for providing essential strains and re- function with default parameters. agents. This work was supported by National Science Foundation EAGER Grant 1548533 (to J.R.P.), National Institutes of Health Grant 5R01GM078153 Other Methods. Growth conditions, plasmids, genetic and transformation (to F.R.C.), The Rockefeller University, and the Stanford University De- methods, and the methods used for identification of the LRE, cross-species- partment of Genetics.

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