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Comparative proteomics reveals timely transport into cilia of regulators or effectors as a mechanism underlying ciliary disassembly Limei Wang, Lixiao Gu, Dan Meng, Qiong Wu, Haiteng Deng, and Junmin Pan J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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Journal of Proteome Research

Comparative proteomics reveals timely transport into cilia of regulators or effectors as a mechanism underlying ciliary disassembly

Limei Wang1, Lixiao Gu2, Dan Meng3, Qiong Wu1, Haiteng Deng2, Junmin Pan1,4*

1

MOE Key Laboratory of Protein Sciences, Tsinghua-Peking Center for Life Sciences,

School of Life Sciences, Tsinghua University, Beijing, China 2

MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University,

Beijing 100084, China 3

Tianjin Key Laboratory of Food and Biotechnology, School of Biotechnology and

Food Science, Tianjin University of Commerce, Tianjin 300134, China 4

Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for

Marine Science and Technology, Qingdao, Shandong Province, China

Author for correspondence: *Junmin Pan, [email protected]; Tel/Fax.: 86 10 62771864

Running title: Comparative proteomics of disassembling cilia

Key

words:

Comparative

proteomics,

Cilia

and

flagella,

Chlamydomonas,

Intraflagellar transport (IFT), Ciliary disassembly Abbreviations: IFT, intraflagellar transport; Microtubule, MT; NaPPi, sodium pyrophosphate

1

ACS Paragon Plus Environment


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ABSTRACT Primary cilia are assembled and disassembled during cell cycle progression. During ciliary disassembly, ciliary axonemal microtubules (MTs) are depolymerized accompanied with extensive posttranslational protein modifications of ciliary proteins including protein phosphorylation, methylation and ubiquitination. These events are hypothesized to involve transport of effectors or regulators into cilia at the time of ciliary disassembly from the cell body. To prove this hypothesis and identify new proteins involved in ciliary disassembly, we analyzed disassembling flagella in Chlamydomonas using comparative proteomics with TMT labeling. 91 proteins were found to increase more than 1.4 fold in four replicates. The proteins of the IFT machinery not only increase but also exhibit stoichiometric changes. The other proteins that increase include signaling molecules, chaperones, proteins involved in microtubule dynamics or stability. Of particular, we have identified a ciliopathy protein C21orf2, the AAA-ATPase CDC48 that is involved in segregating polypeptides from large assemblies or cellular structures, FAP203 and FAP236 that are homologous to stabilizers of axonemal microtubules. Our data demonstrate that ciliary transport of effectors or regulators is one of the mechanisms underlying ciliary disassembly. Further characterization of the proteins identified will provide new insights into our understanding of ciliary disassembly, and likely ciliopathy.

2


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INTRODUCTION Eukaryotic flagella or cilia are MT-based cellular organelles that function in cell motility and signaling. Defects in ciliary assembly and maintenance or signaling are associated with a cohort of diseases termed ciliopathies

1, 2

. However, cilia are

dynamic structures. Cilia undergo disassembly prior to cell division and ciliary disassembly may also occur during cell differentiation or in response to stress 3. The importance of ciliary disassembly is underscored by the finding that proper G1 to S transition depends on ciliary disassembly 4-6. Cancer cells are usually associated with loss of cilia, which may be associated with rapid cell proliferation 7. Ciliary disassembly is triggered by signaling cascades in response to external or internal cues 3. Activation of aurora kinases is the key for ciliary disassembly. CALK, a Chlamdyomonas aurora-like kinase, was initially found to be phosphorylated and required for ciliary disassembly

8-10

. Subsequently, it has been shown that in

mammalian cells HEF1 mediated aurora-A activation phosphorylates HDAC6 to deacetylate axonemal MTs leading to ciliary disassembly

11

. Increases of aurora-A

transcription or activation of aurora-A by CAM, Tricoplein, Plk1, PIFO and CPAP eventually leads to ciliary disassembly 12-18. The deacetylated MTs are expected to be depolymerized by microtubule depolymerizing kinesins. Crkinesin13 and its homologue KIF2A are implicated in ciliary disassembly in Chlamdyomonas and mammalian cells, respectively

19, 20

. These data demonstrate that the ciliary

disassembly mechanisms are conserved at least partly from algae to mammals. Ciliary disassembly occurs from the ciliary tip and the disassembled products are

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transported to the cell body for reutilization

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21, 22

. IFT proteins undergo several fold

increase during ciliary disassembly and are expected to carry back the disassembled ciliary proteins

23, 24

. As cilia contain several hundreds of proteins

25-28

, ciliary

disassembly must involve removal of protein complexes that are associated with axonemal MTs and ciliary membranes. In particular, in motile cilia, axonemal microtubules are tightly associated with dynein arms, radial spokes and additional structures or complexes, which affect axonemal MT stability and ciliary length

29, 30

.

During ciliary disassembly, the ciliary proteins undergo protein modifications. In addition to deacetylation of axonemal microtubules, posttranslational modifications including

protein

disassembling cilia

phosphorylation, 10, 11, 31-33

methylation

and

ubiquitination

occur

in

. Responsible enzymes that are present in the flagella

could be activated during flagellar shortening to exert their function. Alternatively, the responsible enzymes or regulators would transport to cilia for these modifications, which mediate disassembly of ciliary protein complexes or their transport to the cell body. This hypothesis is supported by studies where IFT proteins and MT depolymerizing CrKinesin-13 are increased in the cilia upon induction of ciliary disassembly

19, 23

. Certainly, we could not exclude the possibility that the responsible

enzymes that are present in the flagella could be Chlamydomonas reinhardtii is a widely used model organism for ciliary studies. Its flagella can be easily isolated and has been used for various proteomic studies 25, 31, 34

.The induction of flagellar disassembly can be experimentally manipulated 22, and

the mechanism of ciliary disassembly induced in vitro and under physiological

4


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conditions are conserved

3, 10

.To identify proteins that are transported into cilia during

ciliary disassembly and learn more about the mechanism of ciliary disassembly, Chlamydomonas cells were treated with sodium pyrophosphate (NaPPi) to induce flagellar disassembly. The proteins from control and disassembling flagella were labeled with TMT for comparative proteomic analysis. We have not only identified new proteins that are potentially involved in ciliary disassembly but also revealed that IFT motors and IFT complexes undergo stoichiometric changes.

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MATERIALS AND METHODS Strains and Cell culture Wild type C. reinhardtii strain 21gr (CC-1690) is available from Chlamydomonas Genetic Center (University of Minnesota, St. Paul, MN). Cells were grown in M medium with aeration in a 14:10 h light-dark cycle at 22oC 35. Flagellar isolation and Differential interference microscopy (DIC) To induce flagellar shortening, cells were treated with NaPPi (pH 7.2) for 15 min at a final concentration of 20 mM followed by flagellar isolation. At this time flagellar shortening was barely visualized though flagellar disassembly was induced. Flagella were deflagellated by pH shock and purified essentially as described 35. Briefly, the pH of the cell cultures was rapidly lowered to pH4.5 by drop-wise addition of 0.2 M acetic acid and subsequently neutralized by addition of 0.2 M KOH after 30 seconds. The cell bodies were removed by centrifugation at 600xg for 10 min at 4 oC (himac CF12RX, HITACHI, swing rotor T3S51). Ice-cold 25% sucrose in 10 mM Tris, pH7.2, was added to the flagellar fractions to yield 7% sucrose at final concentration. The samples were then underlayed with 25% sucrose and centrifuged at 1880xg for 10 min at 4 oC. The upper phase that contains purified flagella was further centrifuged at 10,000xg for 10 min at 4 oC (rotor T11A33). Flagella pellets were resuspended in HMDEK buffer (50 mM HEPES [pH 7.2], 5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, 0.1% NP-40). A small portion of flagella isolated before solubilization in buffer were fixed in 1% glutaraldehyde for DIC microscopy. DIC images were captured with Zeiss microscope (AXIO) (Zeiss, Germany) equipped with a

6


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QuantEM:512SC camera (Photometrix, US). Protein digestion and TMT Labeling A total of four replicates were analyzed. For two replicates, 200 µg of flagellar proteins from control and disassembling flagella were resolved respectively by SDS-PAGE followed by commasiee staining. 12 gel slices from each flagellar sample were reduced with 25 mM dithiothreitol and alkylated with 55 mM iodoacetamide in 50 mM disodium phosphate. For another two replicates, 200 µg of isolated flagella were dissolved in PBS containing 8 M urea. After brief sonication, the lysates were centrifuged at 12,000 rpm for 30 min at 4oC (Eppendorf 5417R). The supernatants were collected for determination of protein concentration using a Bicinchoninic acid protein assay kit. 100 µg of proteins were reduced with 5 mM dithiothreitol and alkylated with 12.5 mM iodoacetamide in 50 mM disodium phosphate. Sequencing grade trypsin was used for in gel digestion overnight at 37℃ . Peptides were extracted twice with 0.1% formic acid in 50% acetonitrile aqueous solution for 30 min followed by concentration and drying with a speedvac. Peptides were suspended in 100 µl of 200 mM triethyl ammonium bicarbonate and labeled with TMT label reagent for 1 h at room temperature. Peptides from control and disassembling flagella were labeled by TMT6-126 and TMT6-129, TMT6-129 and TMT6-126, TMT6-127 and TMT6-130, or TMT6-128 and TMT6-131, respectively. The reaction was quenched by 5% hydroxylamine for 15 min. The samples were then desalinated, dried, suspended in 0.1% formic acid and analyzed by nano-LC−MS/MS.

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LC-MS/MS Analysis Samples were separated by a 120 min gradient elution at a flow rate of 0.25 µl/min in a nano-HPLC system (Proxeon, Denmark), which was directly interfaced with a Thermo Q Exactive Mass Spectrometer (for in-gel digestion samples) or with a Thermo Orbitrap Fusion Lumos Mass Spectrometer (for urea treated samples). The analysis was essentially as previously described

36

. The analytical column was a

fused silica capillary column (75 µm ID, 150 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 Å, 5 µm, Varian, Lexington, MA). 0.1% formic acid was used for the mobile phase A, and 100% acetonitrile and 0.1% formic acid was used for mobile phase B. The mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.0.7 software. The parameters for QE and Orbitrap spectrometers were used as the following, respectively: A single full-scan mass spectrum in the Orbitrap, 400−1800 m/z, 70000 resolution or 350-1550 m/z, 120000 resolution; 30% or 38% normalized collision energy (HCD); 2 or 0.7 Da for the mass window for precursors ion selection; 20 or 15s for dynamic exclusion duration; 17,500 or 30,000 for resolution for HCD spectra. The MS/MS spectra from each LC−MS/MS run were searched against the Chlamydomonas fasta database downloaded from Phytozome using an in-house Sequest HT Algorithm in Proteome Discoverer software (version 1.4). The following search criteria were used: requiring full tryptic specificity; allowing one missed cleavage; carbamidomethylation (C) and TMT sixplex (K- and N-terminal) were set as the fixed modifications; the oxidation (M) was set as the variable modification; precursor ion mass tolerances were set at 20 or 10 ppm for all MS acquired in an orbitrap mass analyzer; and the fragment ion mass tolerance was set at 20 mmu for all MS2 spectra. Peptide spectral matches (PSMs) were validated using the Percolator provided by Proteome Discoverer software based on q-values at a 1% false discovery rate (FDR). A peptide whose sequence is only assigned to a given protein group was considered as unique. The FDR was also set at 0.01 for protein identifications. Relative protein quantification was carried out using Proteome

8


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Discoverer software (Version 1.4) following manufacturer’s instructions on the reporter ion intensities per peptide. Proteins with at least two unique peptides were regarded as confident identifications and were further quantified. Protein ratios were calculated as the median of all peptide hits belonging to a protein. Gene cloning and Transformation The full-length gene sequences for FAP109, FAP252 and CDC48 were cloned by PCR from genomic DNA with approximately 1.6, 1.8 and 1.6 kb fragments, respectively, upstream of the start codon. CDC48 was tagged with YFP at the 3’ end while FAP109 and FAP252 were tagged with a 3×HA tag at the 3’ end. The HA-tags were followed by a LC8 terminator cloned from plasmid pKL-3×HA 37 and the YFP tag was followed by a PSAD terminator

38

. The resulting constructs were cloned into a

modified vector pHyg3 harboring a hygromycin B resistance gene 39. All the constructs were verified by DNA sequencing. The final constructs were linearized with ScaI-HF (for HA-tagged constructs) or AfIII (for YFP-tagged construct) and transformed into wild type cells using electroporation 40. Primary antibodies The following primary antibodies were used for immunoblotting (IB) or immunofluorescence (IF) as indicated in the text. Rabbit anti-CALK (1:10,000, for IB) 8; Mouse monoclonal anti-α-tubulin (Sigma-Aldrich;1:200 for IF and 1:3000 or 1:10,000 for IB); Mouse anti-GFP (1:1000 for IB, AbMart); Rat monoclonal anti-HA (clone 3F10, Roche;1:50 for IF and 1:3000 for IB); Mouse monoclonal anti-IC2 (Sigma-Aldrich; 1:20,000 for IB); Mouse monoclonal anti-IFT139 (1:10,000 for IB) and mouse monoclonal anti-IFT81 (1:1000 for IB) (kindly provided by Dennis Diener and Joel

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Rosenbaum, Yale University) 41. SDS-PAGE, Immunoblotting and IF microscopy For these assays, it was essentially as described previously

35

. For IF, the

secondary antibodies used were Texas Red-conjugated goat anti-mouse IgG (Molecular Probes, 1:200) and Alexa Fluor 488-conjugated goat anti-rat IgG (Molecular Probes, 1:200). The samples were viewed on a Zeiss LSM780 META Observer Z2 Confocal Laser Microscope with a 100x oil lens. Images were obtained and processed by ZEN 2009 Light Edition (Zeiss, Germany), Photoshop and Illustrator software (Adobe, USA).

10


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RESULTS AND DISCUSSION Flagellar isolation, sample processing and data analysis We have used NaPPi to induce flagellar disassembly. In the presence of NaPPi, Chlamydomonas cells shorten their flagella gradually almost in a linear fashion and complete disassembly takes about 3 hrs

10, 22

. The mechanism of NaPPi in inducing

flagellar shortening is not clear, it likely involves calcium regulation as NaPPi is a calcium chelator

22

. To avoid issues arising from short flagella for comparative

proteomic analysis, flagella were isolated from cells that were treated with NaPPi for only 15 min when changes of flagellar length were barely detected. To ensure that flagellar disassembly process was indeed triggered, cells that were treated with NaPPi were analyzed for CALK phosphorylation. CALK underwent a molecular weight shift, an indication of CALK phosphorylation and initiation of flagellar disassembly 8, in the NaPPi treated cells but not in control cells, indicating flagellar disassembly was induced (Fig. 1 A). Isolated flagella were examined microscopically and found to contain pure flagella (Fig. 1B). As IFT proteins increase in the flagella during flagellar shortening 10, 23

, the isolated flagella were examined for IFT increase. Both IFT-A protein IFT139

and IFT-B protein IFT81 were indeed increased in disassembling flagella (representative data from two experiments) (Fig. 1C). Thus, the above analysis shows that NaPPi treatment triggered flagellar disassembly and the flagellar samples were suitable for further analysis. A total of four replicates were analyzed. Flagellar proteins from two replicates

11



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were resolved in 10% acrylamide gel for SDS-PAGE followed by coomassie blue staining (Fig. 1D). The gels were cut into 12 slices followed by trypsin digestion. Flagellar proteins from another two replicates were treated with 8 M urea followed by trypsin digestion. The peptides were analyzed by LC-MS/MS. A flow chart for the analysis is shown in Fig. 1E. The identified peptides were searched in Chlamydomonas genome database (V5.5) for protein identification. Proteome Discoverer Software (version 1.4) (ThermoFisher) was used for relative protein quantification.

Comparative analysis of protein fold changes in disassembling flagella Mass spectrometric data from four replicates were analyzed. A total of 452 proteins that were identified by two or more unique peptides were found (Table S1). The number of proteins identified is comparable to a previous publication

25

. To find

proteins that exhibit significant changes before and after inducing flagellar disassembly, proteins that showed more than 1.4 fold changes with p value less than 0.05 were considered. By this criterion, a total of 91 proteins showed increase in disassembling flagella while none proteins showed decrease. The information for these increased proteins is presented in Table S2. Validation of protein level increases in disassembling flagella Previous biochemical studies show that several proteins increase in the flagella during flagellar disassembly. CrKinesin13, a microtubule depolymerizer, increases in disassembling flagella to promote disassembly of axonemal microtubules

19, 42

. In

12


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addition, IFT proteins IFT139 and IFT172 were also increased

23

. In our proteomic

data, we found that CrKinesin 13 increased 2.82 fold in average, and IFT139 and IFT172 increased 1.91 and 1.70 fold in average, respectively (Table S2). To further validate our data, we have selected FAP109 and FAP252 that exhibited 1.52 and 1.74 fold increase, respectively, for additional analysis (Table S2). The genes encoding these two proteins were tagged respectively with 3xHA and transformed into Chlamydomonas cells. Immunostaining of positive transformants with anti-HA antibody showed that these proteins were located in the flagella, indicating they are bona fide flagellar proteins (Fig. 2A). To determine whether they are increased in disassembling flagella, the cells were treated with NaPPi. Immunostaining did not reveal significant increase of these proteins in disassembling flagella (data not shown). This may be due to the limitation of the resolution of the immuofluorescence technique. Flagella were then isolated from cells before and after NaPPi treatment followed by immunoblot analysis (Fig. 2B). As expected, both proteins increased in disassembling flagella. Thus, our comparative proteomic data can provide reliable resource for identifying new proteins that increase in disassembling flagella and likely function in flagellar disassembly. We have examined the phenotypes of these two transgenic strains as well as CDC48 transgenic strains (see below), we did not find any changes in flagellar length and flagellar disassembly compared to the wild type cells. It is likely because that the levels of the transgenic expression were relatively low.

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Distinct groups of proteins show increase in disassembling flagella The proteins that showed significant increase in disassembling flagella were categorized into different groups (Fig. 3, Table S2). These include IFT proteins, MT-associated proteins, signaling molecules, chaperones, other known flagellar proteins, proteins involved in metabolism and transcription/translation, and other proteins without known functions. The increase of a large number of proteins into flagella during flagellar disassembly indicates that the cells need to deliver proteins into the flagella to mediate the flagellar disassembly processes. Flagellar disassembly is complex. It involves signaling generated by internal or external cues that trigger flagellar disassembly, depolymerization of axonemal microtubules, disassembly of large protein structures such as dynein arms and radial spokes, and returning disassembled components to the cell body 3. It is expected that molecules that function in these different processes are transported to the flagella to exert their function during flagellar disassembly. However, it should be cautioned that some proteins identified may not necessarily be involved in flagellar disassembly per se. Some proteins that are increased may be due to cellular stress caused by NaPPi treatment. Furthermore, contamination of cell body proteins may not be completely excluded. Nonetheless, as NaPPi indeed induces flagellar disassembly, some proteins that were identified are expected to mediate flagellar disassembly.

Analysis of IFT protein changes reveals stoichiometric regulation of IFT

14


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IFT proteins represent the major group of proteins that increase in disassembling flagella (Fig. 3). Though most of the IFT proteins in all four replicates were identified by two or more unique peptides, some of them were found in two or three replicates with identification by two or more peptides (Table 1). The increase of IFT proteins provides an opportunity to examine possible IFT regulation during flagellar disassembly. During anterograde IFT, kinesin-II motor carries IFT-A and IFT-B complexes as well as cytoplasmic dynein 1/2b from ciliary base to the tip. After remodeling of IFT complexes and motors at the ciliary tip, cytoplasmic dynein 1/2b moves IFT complexes back to the cell body 43, 44. The three subunits of kinesin-II increase 2.22± 0.11 (mean±STD) fold in average. The ratio of FLA8/FLA10/KAP is 1.0/1.1/1.1, indicating that kinesin-II forms a stable complex and its composition does not change during flagellar disassembly. The average increase of IFT-B proteins is 1.96±0.15 fold in average (only proteins that were identified by two or more peptides in three replicates were taken into account). Compared with the average increase of IFT-B, IFT52 has a 14% increase. These data suggest that IFT-B complex might undergo stoichiometric changes during flagellar disassembly. The increase of all IFT-A components except for IFT43 is very close with 1.86± 0.05 in average. The ratio of IFT144/IFT140/IFT139/IFT122/IFT121 is close to 1, indicating these five subunits form a tight complex. Intriguingly, IFT43 increased 2.23 fold and a 20% increase over the rest of the IFT-A components. These data are consistent with a recent report where IFT43 plays a unique role in IFT regulation

15



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among other IFT-A proteins 45. IFT dynein (cytoplasmic dynein 1/2b) complex is comprised of IFT dynein heavy chain, light intermediate chain, intermediate chains and light chains

46

. All the

components of IFT dynein were identified. As LC8, LC7b and Tctex2b are also part of axonemal dynein arms

47-50

, the ratio of the rest of the components of IFT dynein is

calculated. The ratio of DHC1b/D1bLIC/D1bIC1/D1bIC2/Tctex1 is 1.0/1.4/1.0/0.9/0.7. The large increase of D1bLIC and decrease of Tctex1 relative to IFT dynein heavy chain indicates that IFT dynein complex also undergoes stoichiometric changes during flagellar disassembly. D1bLIC has been shown to play a role in binding of ciliary cargos

51, 52

. The vast increase of D1bLIC is consistent with its role in

transporting ciliary proteins that are disassembled during ciliary disassembly. Our data demonstrate that IFT-trafficking is increased during flagellar disassembly, which is consistent with previous finding

23

. The increase of IFT

trafficking coupled with flagellar disassembly reflects the need for the cells to transport disassembled products into the cell body during vigorous flagellar disassembly. In addition, our data also provide the first evidence that the stoichiometry within IFT complex and IFT dynein may undergo changes.

Changes of axonemal MT associated proteins during flagellar disassembly Axonemal MTs are major structures of cilia. During ciliary disassembly, axonemal microtubules are depolymerized through the action of microtubule depolymerizing CrKinesin-13 in Chlamydomonas and its homologue KIF2A in mammalian cells

19, 20

.

16


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Indeed, CrKinesin-13 was found to increase 2.82 fold in average (Table 2). In mammalian cells, microtubule deaceylase HDAC6 is phosphorylated by aurora-A and required for ciliary disassembly 11. As aurora-A is activated at the basal body region, the phosphorylated HDAC6 is expected to be transported to cilia for deacetylation of axonemal microtubules though it has not been shown. We have found two deacetylases (Cre11.g467785 and Cre11.g467706) in the flagellar proteome, which are most closely related to HDAC6 (GenBank: EAW59745.1) in the human proteome. The e-values for HDAC6 are 1e-80 and 2e-74, respectively, when Cre11.g467785 and Cre11.g467706 were blasted in the human proteome, suggesting that Cre11.g467785 is likely an orthologue of human HDAC6. These two proteins were identified in at least three replicates. However, they were identified by two or more unique peptides only in two replicates. Cre11.g467785 and Cre11.g467706 increase 1.74 and 1.45 fold, respectively, in disassembling flagella. These data suggest that Chlamdyomonas HDAC6 homologues likely increase in disassembling flagella to mediate deactylation of axonemal microtubules. We have also found other proteins that are involved in MT dynamics (Table 2). EB1 localizes to the tip of growing and steady state flagella

53, 54

. Though it is a

MT-plus end tracking protein, it also associates with the flagellar tip during flagellar shortening. The finding of the increase of EB1 indicates that MT dynamics mediated by EB1 may be required for disassembly of axonemal microtubules. Another possibility is that EB1 may be involved in IFT turnaround at the flagellar tip

54

.

FAP194 (PF16) localizes to the C1 microtubule and its null mutation results in

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loss of C1 microtubule from the axoneme, indicating it regulates C1 microtubule stability

55

. Thus, PF16 increases may reflect a feedback control mechanism during

flagellar disassembly. SAXOs, stabilizers of axonemal microtubules, are present only in ciliated or flagellated organisms 56. Human SAXO1 stabilizes MTs in vitro and is present in cilium. Depletion of SAXO1 reduces ciliary length likely through its role in MT stability

57

.

Three homologues of SAXOs are present in Chlamydomonas that include FAP203, FAP236 and FAP257. We have found that FAP203 and FAP236 were both increased in disassembling flagella. Their increase in disassembling flagella is intriguing. One speculation is that these microtubule stabilizers may undergo posttranslational modifications to switch its activity toward microtubule disassembly. Another possibility is that it may reflect a feedback mechanism. Future work is needed to determine how they participate in flagellar stability and disassembly.

Flagellar increase of signaling molecules Ciliary proteins undergo posttranslational modifications during ciliary disassembly 3

. The responsible enzymes are expected to be regulated by signaling events. Several

signaling molecules or their regulators including protein kinases, phosphatases, GTPase and phosphodiesterase were increased (Table 3). Interestingly, a Rab GDP dissociation inhibitor was also increased. As it is involved in membrane trafficking 58, it is likely involved in ciliary membrane removal during ciliary disassembly.

18


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CDC48 and C21orf2 We have found that CDC48 (also known as p97) was increased 2.64 fold (Table 3). Immunoblotting of cells expressing CDC48GFP found that CDC48 was indeed increased in disassembling flagella (Fig. 2C). CDC48 is a conserved AAA-ATPase and functions to segregates ubiquitinated molecules from protein complexes and cellular structures. CDC48 is directed to its target by an array of different adaptors. The released polypeptides can be degraded by the ubiquitin proteasome system or recycled

59

. Recently, it has been shown that CDC48 and its adaptor UBXN10 are

required for ciliogenesis likely through regulation of the stability of IFT-B complex formation though CDC48 is not found in the cilium

60

. The protein ubiquitination

system is present in Chlamdyomonas flagella and protein ubiquitination is increased in disassembling flagella 33. As CDC48 targets ubiquitinated proteins and is increased in disassembling flagella, it indicates that CDC48 likely functions in ciliary disassembly. As UBXN10 does not have the ubiquitin-associated domain for recognizing polyubiquitin chains on substrates

60

, other adaptors for CDC48 must be involved to

mediate flagellar disassembly. C21orf2 is a human disease gene involved in ciliopathies characterized with retinal and skeletal impairment and localized to cilia inhibits ciliogenesis

61, 62

. RNAi deletion of C21orf2

62

. However, its working mechanism is not known. The finding of

its increase in disassembling flagella hints that it may function in ciliary stability (Table 3).

19



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Page 20 of 35

Other proteins that increase in disassembling flagella Earlier flagellar proteome analysis has identified multiple uncharacterized proteins, termed FAPs (flagella-associated proteins)

25

. Several FAPs were increased

in disassembling flagella (Table 3), their role in flagellar disassembly remains to be identified. Protein methylation occurs during flagellar disassembly 32. Consistently with this, we have identified increase of methionine synthase (MetE) in disassembling flagella (Table 3). We have also identified chaperones, proteins involved in metabolism and transcription and translation (Table S2). Protein chaperones regulate protein folding and unfolding 63. During flagellar disassembly, large protein complexes are disassembled and protein folding and unfolding is expected to be involved. For example, HSP90, which increases 2.75 fold in disassembling flagella, is known to form a complex with HDAC6, which is a microtubule deacetylase and required for ciliary disassembly

11, 64

. Proteins involved in transcription/translation were also

indentified in flagellar proteome previously

25

. Whether these proteins are

contaminants or actually function in flagella remain to be defined.

Conclusion We have used a comparative proteomic approach to study the mechanism of ciliary disassembly. The finding that multiple proteins increase in disassembling flagella suggests that timely transport of regulators or effectors into cilia is one of the mechanisms for ciliary disassembly. The proteins that are increased in disassembling flagella are expected to be transported by IFT. We have found that the IFT machinery

20


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is upregulated in disassembling flagella and IFT complex and motors undergo stoichiometric changes. Thus, one future area research is study how these proteins are “activated” and loaded on IFT complexes, how IFT trafficking is controlled and how stoichiometry of IFT complex and motors is regulated. Further study of the axonemal MT-associated proteins, signaling molecules and especially CDC48 will provide new insights into the mechanism of ciliary disassembly. Lastly, the identification of human disease causing gene C21orf2 underscores the importance of this study in human health and disease and will contribute to our understanding of C21orf2 related ciliopathy.

21



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Page 22 of 35

Supporting Information The supplemental materials include the following: Table S1, Proteins identified by two or more unique peptides in all four replicates; Table S2, Up-regulated proteins in disassembling flagella compared to control.

ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (31330044, 31671387),

National

Basic

Research

Program

of

China

(973

program)

(2013CB910700) and Sino-Germany Science Center (GZ990) (to J. P.).

22


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encodes a protein that localises to the photoreceptor primary cilium. Br J Ophthalmol 2015, 99 (12), 1725-31. (62) Wheway, G.; Schmidts, M.; Mans, D. A.; Szymanska, K.; Nguyen, T. M.; Racher, H.; Phelps, I. G.; Toedt, G.; Kennedy, J.; Wunderlich, K. A., et al., An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nat Cell Biol 2015, 17 (8), 1074-87. (63)Zhang, X.; Beuron, F.; Freemont, P. S., Machinery of protein folding and unfolding. Curr Opin Struct Biol 2002, 12 (2), 231-8. (64) Boyault, C.; Zhang, Y.; Fritah, S.; Caron, C.; Gilquin, B.; Kwon, S. H.; Garrido, C.; Yao, T. P.; Vourc'h, C.; Matthias, P., et al., HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates. Genes Dev 2007, 21 (17), 2172-81.

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Table 1. Increase of IFT proteins in disassembling flagella Complex

Protein

Complex

Protein

IFT-B

IFT172 IFT88 IFT81 IFT80 IFT74 IFT70

1.70 1.88 1.93 1.77 1.76 1.97

IFT-A

IFT144 IFT140 IFT139 IFT122 IFT121

IFT57

1.97 1.86 1.97

Kinesin-II

FLA10 FLA8 KAP

2.11 2.33 2.22

2.24 2.01 1.97 2.17 1.73 2.08 2.12

IFT-dynein

DHC1b D1bLIC D1bIC1 D1bIC2

2.42 3.31 2.39 2.27 1.98 1.66 1.80 1.77

IFT56 IFT54

b

IFT52 IFT46 IFT38

b

b

IFT27 a IFT25 IFT22 IFT20

b

Fold increase

IFT43

b

a

LC7b LC8 Tctex1 Tctex2bb

Fold increase 1.88 1.78 1.91 1.86 1.88 2.23

Proteins were identified by two or more unique peptides in two (a), three (b) or four replicates (the rest of the proteins).

28


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Table 2. Increase of axonemal MT associated proteins Name CrKinesin13 EB1 FAP203 FAP236 FAP194

Description Microtubule depolymerase Microtubule plus-end binding protein Stabilizer of axonemal MTs Stabilizer of axonemal MTs PF16, C1 microtubule protein

Ave. fold increase 2.82 1.88 1.90 1.70 1.64

29



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Table 3. Increase of signaling molecules and flagella-associated proteins Gene ID

Name

Description

Ave. fold of increase

Signaling molecules Cre12.g493250

PKG

cGMP dependent protein kinase

1.99

Cre13.g564500

CDPK11

Calcium dependent protein kinase

1.51

Cre19.g750597

MAPK5

MAP kinase 5

1.87

Cre12.g528000

AMPK

AMP-activated protein kinase

2.66

Cre07.g325724

PTP1

Protein tyrosine phosphatase

1.75

Cre06.g292550

PP1A

Serine/threonine phosphatase

2.32

Cre09.g388467

GDI

Guanine nucleotide dissociation inhibitor

2.49

Cre02.g104900

FAP204

Containing a YCHF GTPase domain

2.18

Cre06.g257500

FTT2

14-3-3 family protein

2.21

Cre12.g559250

FTT1

14-3-3 family protein

2.05

Cre07.g342350

PDE14

3',5'-cyclicnucleotidephosphodiesterase

1.45

Flagella-associated proteins Cre06.g269950

CDC48

AAA-ATPase

2.64

Cre14.g625550

C21orf2

Leucine rich repeat-containing protein

1.59

Cre03.g180750

METE

Cobalamin-independent

2.05

Cre17.g717150

FAP263

Homologous to HsCCDC113

1.79

Cre16.g650550

FAP103

Nucleoside diphosphate kinase-like

2.06

Cre17.g718900

FAP88

Unknown function

2.65

Cre01.g015850

FAP68

Unknown function

2.08

Cre16.g679550

FAP277

Serpini1 protein

1.97

Cre10.g466650

FAP252

EF hand containing protein

1.74

Cre01.g012800

FAP230

Unknown function

1.68

Cre10.g450450

FAP18

Serine endopeptidase

1.53

Cre09.g395769

FAP177

Unknown function

1.88

Cre17.g735350

FAP164

Ankyrin repeats protein

1.52

Cre12.g536100

FAP126

Similar to 60S ribsomal protein L3 like

1.57

Cre07.g321400

FAP113

Unknown function

1.86

Cre06.g256450

FAP119

C1a-34,

methionine

synthase

central

pair

microtubule

1.90

pair

microtubule

2.07

projection protein Cre09.g389282

FAP114

C1a-32,

central

projection protein Cre01.g034550

FAP109

EF hand containing protein

1.52

30


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Figure legends Figure 1. Falgellar sample collection and sample processing. (A) Immunoblot analysis of CALK from cells treated with NaPPi for 15 min to induce flagellar disassembly. Molecular weight shift indicates CALK phosphorylation. (B) DIC microscopy of isolated flagella. Bar, 10 µm. (C) IFT increase in disassembling flagella. Flagella isolated from cells treated with NaPPi or not were analyzed by immunoblotting with the antibodies as indicated. Representative data from two experiments are presented. (D) 200 µg total flagellar proteins from control or NaPPi treated cells were resolved on SDS-PAGE followed by coomassie blue staining. The gels were each cut into12 slices for mass spectrometry analysis as detailed in Methods. For another two replicates, flagellar proteins were treated with 8 M urea followed by trysin digestion and analysis. (E) Flow chart of sample processing and analysis.

Figure 2. Validation of increase of identified proteins in disassembling flagella. HA-tagged FAP109 and FAP252, or GFP-tagged CDC48 were expressed in Chlamydomonas cells. (A) Immunostaining with anti-HA antibody shows that FAP109 and FAP252 are flagellar proteins. Bar, 10 µm. Increase of FAP109 and FAP252 (B) or CDC48 (C) in disassembling flagella shown by immnoblotting. Flagella isolated from cells before and after NaPPi treatment were analyzed by immunoblotting. Anti IC2 or α-tubulin antibodies were used as loading control.

Figure 3. Grouping of proteins that were increased in disassembling flagella. A total of 91 proteins identified by two or more peptides in four replicates were analyzed. 31



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Figure 1. Falgellar sample collection and sample processing. (A) Immunoblot analysis of CALK from cells treated with NaPPi for 15 min to induce flagellar disassembly. Molecular weight shift indicates CALK phosphorylation. (B) DIC microscopy of isolated flagella. Bar, 10 µm. (C) IFT increase in disassembling flagella. Flagella isolated from cells treated with NaPPi or not were analyzed by immunoblotting with the antibodies as indicated. Representative data from two experiments are presented. (D) 200 µg total flagellar proteins from control or NaPPi treated cells were resolved on SDS-PAGE followed by coomassie blue staining. The gels were each cut into12 slices for mass spectrometry analysis as detailed in Methods. For another two replicates, flagellar proteins were treated with 8 M urea followed by trysin digestion and analysis. (E) Flow chart of sample processing and analysis. 120x172mm (300 x 300 DPI)


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Figure 2. Validation of increase of identified proteins in disassembling flagella. HA-tagged FAP109 and FAP252, or GFP-tagged CDC48 were expressed in Chlamydomonas cells. (A) Immunostaining with anti-HA antibody shows that FAP109 and FAP252 are flagellar proteins. Bar, 10 µm. Increase of FAP109 and FAP252 (B) or CDC48 (C) in disassembling flagella shown by immnoblotting. Flagella isolated from cells before and after NaPPi treatment were analyzed by immunoblotting. Anti IC2 or α-tubulin antibodies were used as loading control. 121x200mm (300 x 300 DPI)



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Figure 3. Grouping of proteins that were increased in disassembling flagella. A total of 91 proteins identified by two or more peptides in four replicates were analyzed. 109x83mm (300 x 300 DPI)


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For TOC Only - Comparative proteomic analysis of up-regulated proteins in disassembling flagella 79x54mm (300 x 300 DPI)


Comparative proteomics reveals timely transport ... - ACS Publications

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