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[…] Proteins of whole sperm, isolated heads, or flagella were resuspended in an SDS sample buffer and loaded on a SDS gel; after proteins had migrated approximately 1 cm into the separation gel, the gel was stained with Coomassie. The single gel band was excised for every sample, and proteins were in-gel digested with trypsin (Promega, Sunnyvale, CA); peptides were separated by RP-LC (180 min gradient 2–85% acetonitrile, (Thermo Fisher Scientific)) using a nanoAcquity LC System (Waters, Milford, MA) equipped with a HSS T3 analytical column (1.8 µm particle, 75 µm x 150 mm) (Waters) and analyzed twice by ESI-LC-MS/MS, using an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with a 300-2,000 m/z survey scan at 240,000 resolution, and parallel CID of the 20 most intense precursors from most to least intense (top20) and from least to most intense (bottom20) with 60 s dynamic exclusion. All database searches were performed using SEQUEST and MS Amanda algorithm (), embedded in Proteome Discoverer (Rev. 1.4, Thermo Electron 2008-2011, Thermo Fisher Scientific), with both a NCBI (26,623 entries, accessed December 20th, 2010) and a Uniprot (40,895 entries, accessed April 24th, 2014) zebrafish sequence protein database, both supplemented with the DrCNGK protein sequence (). Only peptides originating from protein cleavage after lysine and arginine with up to two missed cleavages were accepted. Oxidation of methionine was permitted as variable modification. The mass tolerance for precursor ions was set to 8 ppm; the mass tolerance for fragment ions was set to 0.6 amu. For filtering of search results and identification of DrCNGK, a peptide FDR threshold of 0.01 (q-value) according to Percolator () two unique peptides per protein and peptides with search result rank 1 were required. [...] Alignments for the calculation of the phylogenetic tree were done with ClustalOmega. Tree was depicted with Tree view (). The following ion channel sequences were used for the phylogenetic tree: CNGK channels from zebrafish (Danio rerio, DrCNGK, XP_001335499.5); rainbow trout (Oncorhynchus mykiss, OmCNGK, CDQ79437.1); spotted gar (Lepisosteus oculatus, LoCNGK, W5MTF2); West Indian ocean coelacanth (Latimeria chalumnae, LcCNGK, H3BE11); sea urchin (Arbacia punctulata, ApCNGK); acorn worm (Saccoglossus kowalevskii, SkCNGK, XP_002731383.1); amphioxus (Branchiostoma floridae, BfCNGK, XP_002592428.1); starlet sea anemone (Nematostella vectensis, NvCNGK, XP_001627832); vasa tunicate (Ciona intestinalis, CiCNGK, XP_002123955); sponge (Amphimedon queenslandica, AqCNGK, I1G982); choanoflagellate (Salpingoeca rosetta, SrCNGK, XP_004992545.1); murine HCN channel 1 (Mus musculus, mHCN1, NP_034538), 2 (mHCN2, NP_032252), 3 (mHCN3, NP_032253.1), and 4 (mHCN4, NP_001074661), and the HCN channel from sea urchin (Strongylocentrotus purpuratus, SpHCN1, NP_999729); rat cyclic nucleotide-gated channels CNGA1 (Rattus rattus, rCNGA1, NP_445949), A2 (rCNGA2, NP_037060), A3 (rCNGA3, NP_445947.1), and A4 (rCNGA4, Q64359); the KCNH channels from fruit fly (Drosophila melanogaster, DmEAG, AAA28495) and human (Homo sapiens, hERG, BAA37096.1); murine voltage-gated Nav channels (Mus musculus, mNav 1.1, NP_061203 and mNav 1.6, NM_001077499.2); murine voltage-gated Cav channels (Mus musculus, mCav1.1, NP_055008, mCav2.3, NP_033912.2, and mCav3.1, NP_033913.2); and voltage-gated Kv channels from fruit fly (Drosophila melanogaster, DmShaker, CAA29917.1) and mouse (Mus musculus, mKv3.1, NM_001112739.1). [...] Sperm were loaded for 45 min at room temperature with either 30 µM DEACM-caged cAMP, 30 µM DEACM-caged cGMP, or 40 µM caged Ca2+ (NP-EGTA, Life Technologies). A UV light-emitting diode (365-nm LED; M365L2-C, Thorlabs, Newton, NJ) was used for photolysis of caged compounds. Experiments using caged Ca2+ and DEACM-caged nucleotides were carried out using a UV power of 25 and 22 mW, respectively. Flash duration was 300 ms. Pluronic (0.5%) was added to the incubation solution. Sperm were kept quiescent during incubation in ES solution (292 mOsm x L-1). Swimming was initiated by a hypoosmotic shock diluting sperm 1:20 in an activation solution containing (in mM): NaCl 70, KCl 5.4, MgCl2 1, CaCl2 1.8, glucose 10, and Hepes 5 at pH 7.4 adjusted with NaOH (167 mOsm x L-1). Swimming behaviour was observed with a dark-field condenser in an inverse microscope (IX71, Olympus, Tokio, Japan) with 20x magnification (UPLSAPO, NA 0.75). Movies were recorded at 30 Hz using a back-illuminated electron-multiplying charge-coupled device camera (DU-897D; Andor Technology, Belfast, United Kingdom). Sperm trajectories were tracked using custom-made software written in MATLAB (Mathworks). The software can be made available upon request. The average swimming path (ASP) was calculated by filtering the tracked coordinates with a second degree Savitzky-Golay filter with a 200 ms span. The curvature (κ) of the swimming path was calculated using the formula:bbbκ=x˙y¨−y˙x¨x˙2+y˙23/2, where x and y are the coordinates of the ASP.To assess cAMP loading and release, we recorded the fluorescence increase due to DEACM-OH release after photolysis of DEACM-caged cAMP (). Release and fluorescence excitation was achieved simultaneously using the same UV LED (power 1.75 mW). Light was coupled to the microscope with a dichroic mirror (455DRLP, XF2034, Omega Optical, Brattleboro, VT) and fluorescence was long-pass filtered (460ALP; XF309; Omega Optical). Single cells were recorded at 50 Hz.Data analysis: Statistical analysis and fitting of data were performed, unless otherwise stated, using Sigma Plot 11.0, GraphPadPrism 5, or Clampfit 10.2 (Molecular Devices). All data are given as mean ± standard deviation (number of experiments).Note: All cell lines used in this study will be sent for STR profiling. Mycoplasma testing was performed using the Promokine Mycoplasma Test Kit 1/C (PromoCell GmbH, Heidelberg, Germany). Results of this test can be supplied upon request. […]

Pipeline specifications

Software tools Clustal Omega, GraphPad Prism
Applications Miscellaneous, Phylogenetics
Organisms Danio rerio
Chemicals Calcium, Nucleotides, Cyclic, Potassium