1. Volume 19, Issue 2, Pages 133-139 (January 2009)
Chlamydomonas CAV2 Encodes a Voltage- Dependent Calcium Channel Required for the Flagellar Waveform Conversion 
Kenta Fujiu, Yoshitaka Nakayama, Ayaka Yanagisawa, Masahiro Sokabe, Kenjiro Yoshimura 
Current Biology 
Volume 19, Issue 2, Pages (January 2009)
DOI: /j.cub
Copyright © 2009 Elsevier Ltd Terms and Conditions

2. Figure 1 Genetic characterization of ppr2
(A) RT–PCR of the mRNA of genes with high homology (left) and moderate homology (right) to VDCCs.
(B) Schematic diagram of CAV2 intron-exon map (exon is boxed) of wild-type (WT) and ppr2 genomic DNA. The HindIII-flanked region was recovered from ppr2 genomic DNA and amplified by inverse PCR with primers inv and inv′. The NIT1 insertion identified by the inversed PCR is indicated. Arrowheads indicate primers used for analysis of the genomic DNA.
(C) PCR amplification of the genomic DNA with primer sets e-e′ and f-f′ shown in panel (B).
(D) Genotype analysis of the offspring of the ppr2 × wild-type cross. The NIT1 insertion was detected with primers n and w′, and the corresponding region in the wild-type genome was detected with primers w and w′ (B). The presence or absence of the photophobic response (Photo) or mechanoshock response (Mechano) is indicated by + and −, respectively.
(E–H) Suppression of CAV2 expression by RNAi. (E) Immunoblot of the flagella isolated from RNAi transformants with CAV2 and α-tubulin (ATU) antibodies. The expression level relative to control strain transformed with empty vector (vector) was 12% (A1), 92% (F3), 54% (F2), and 9% (F1). (F) Photophobic response in control (vector, n = 35) and RNAi knockdown strain F1 (CAV2 KD, n = 64). (G and H) Swimming track of the RNAi F1 cells (G) and control cells (H) after photostimulation (arrowhead). The interval between consecutive images is 0.2 s.
Current Biology  , DOI: ( /j.cub )
Copyright © 2009 Elsevier Ltd Terms and Conditions

3. Figure 2 Sequence Analysis of CAV2
(A) Hydrophobicity profile of CAV2 (after Kyte and Doolittle [30]). Brackets and bars represent domains (I to IV) and transmembrane segments, respectively.
(B) Multiple sequence alignment of the pore-lining regions of various Ca2+ and Na+ channels. The species compared in (B) and (C) are Chlamydomonas reinhardtii (Cr), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Nasonia vitripennis (Nv), Discopyge ommata (Do), Mus muscus (Mm), and Homo sapiens (Hs). The accession numbers are listed in Table S1.
(C) Sequence alignments of pore loop and transmembrane regions were used for phylogenetic tree analysis with the RAxML program [31]. Numbers in each tree represent bootstrap scores obtained from 100 bootstrap tests.
Current Biology  , DOI: ( /j.cub )
Copyright © 2009 Elsevier Ltd Terms and Conditions

4. Figure 3 Localization of CAV2
(A) Expression of CAV2 during flagellar regeneration. RNA was collected from cells before the deflagellation (0 min) and 30 min and 45 min after the deflagellation for quantitative real-time RT-PCR analysis. Fructose-bisphosphate aldolase 3 (FBA) and β-tubulin (BTU) were used as a loading control and a positive control, respectively. The expression level relative to that at 0 min is shown. Scale bars indicate standard error of the mean.
(B) Immunoblot of a Chlamydomonas cell body and the flagellar protein probed with CAV2 antibody. Protein from same number of cells was loaded on each lane.
(C) Immunoblot of the flagellar protein of wild-type and ppr mutants probed with CAV2 antibody.
(D) Laser confocal (left) and differential-interference contrast microscope image (right) of a wild-type cell immunolabeled with CAV2 antibody.
(E) A ppr2 cell immunostained with CAV2 antibody. A phase-contrast image is shown on the right.
(F) Double immunofluorescence staining of a wild-type cell with CAV2 (green) and α-tubulin (red) antibodies.
(G) The intensity of the immunofluorescence along the left flagellum shown in panel (F).
(H) Total amount of CAV2 immunofluorescence examined on flagella with various lengths. The line represents linear regression.
(I) Model for the Ca2+ influx in the photophobic (mechanoshock) response (left) and deflagellation (right). The site of Ca2+ influx is shown by arrowheads, and the region with increased Ca2+ concentration is indicated by red.
Current Biology  , DOI: ( /j.cub )
Copyright © 2009 Elsevier Ltd Terms and Conditions

5. Figure 4
Localization of CAV2 in fla10, a Mutant Defective in IFT at Nonpermissive Temperature
(A) A fla10 cell at nonpermissive temperature shown as confocal cross-sections along the longitudinal axis. The cross-section goes from the cell body (i) to the apical region (vi) and is shown as a merged image of α-tubulin (red) and CAV2 immunofluorescence (green).
(B–D) Immunofluorescence of α-tubulin (red) and CAV2 (green) and merged images (right). Samples are fla10 cells at nonpermissive temperature (B), wild-type cells at nonpermissive temperature (C), and fla10 cells at permissive temperature (D). Arrowheads indicate the accumulation of CAV2 at the basal region of flagella.
(E and F) Localization of CAV2 accumulation after deflagellation of fla10 cells preincubated at nonpermissive temperature. The anterior end of the cell body (E) and the detached flagellum (F) are shown. An arrow indicates the proximal end of the flagellum. Scale bars represent 5 μm (A–D) and 2 μm (E and F).
(G) Proportion of cells that have accumulated CAV2 close to the flagellar base. Data for wild-type and fla10 cells at 25°C and 32°C are shown (n = 53–103).
(H) Immunoblot analysis of CAV2 and α-tubulin (ATU) in isolated flagella of wild-type and fla10 cells at permissive and nonpermissive temperatures.
Current Biology  , DOI: ( /j.cub )
Copyright © 2009 Elsevier Ltd Terms and Conditions