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The FA2 gene of Chlamydomonas encodes a NIMA family kinase with roles in cell cycle progression and microtubule severing during deflagellation

Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6

View larger version (34K): [in a new window]   Fig. 8. RNAi of FA2 in wild-type Chlamydomonas cells. (A) Construct for triggering RNAi. The construct pBSFA2-RNAi was created by cloning a cDNA cassette containing exons 1 to 5 in inverse orientation at the 3' end of the corresponding genomic DNA. (B) Northern analysis of FA2 in cells transformed with the pBSFA2-RNAi construct. Each lane contains 10 µg of polyadenylated RNA obtained from a population of asynchronous cells from the strain indicated. The blot was probed with 0.7 kb of FA2 cDNA and re-probed with 0.5kb of CBL as a loading control. (C) Ratio of FA2 mRNA signal compared with that of CBL. (D) Deflagellation response of each cell type to acid. (E) Cell size distribution of RNAi colony number 17. One hundred cells were measured.

View larger version (70K): [in a new window]   Fig. 1. Nucleotide and deduced amino-acid sequence of FA2. Nucleotides are numbered on the left and amino acids on the right. The putative ATP-binding site is underlined and the putative kinase site is shaded. The sequence of FA2 has been submitted to GenBank under the Accession No. AF479588.

View larger version (116K): [in a new window]   Fig. 2. Amino-acid sequence comparison of Fa2p and other NIMA kinases. The alignment was generated using Clustal W and Genedoc using gaps to maximize homology. Black, dark grey and light grey shading represents 100%, 80% and 60% conservation, respectively. The numbers indicate the relative positions of the amino acids from the N-termini of the various proteins. Sequences used: Homo sapiens STK2 (XP_003216.2), Homo sapiens Nek2 (NP_002488), Mus musculus STK2 (JC7122), Mus musculus Nek1 (P51954), Mus musculus Nek2 (NP_035022), Mus musculus Nek3 (Q9R0A5) and Mus musculus Nek4 (NP_035979).

View larger version (54K): [in a new window]   Fig. 3. Northern analysis of FA2 gene and the fa2 mutant alleles. (A) Schematic representation of the mutant alleles. fa2-1, fa2-3 and fa2-4 were generated by NIT1 insertion and fa2-2 was generated by UV mutagenesis (see Finst et al., 1998). (B) Ten micrograms of polyadenylated RNA from log phase cells was loaded per lane. The blot was probed with 0.7 kb of FA2 cDNA, and re-probed with 0.5 kb of the CBL gene as a loading control. (C) Northern analysis of RNA from wild-type cells following deflagellation by pH shock (Witman et al., 1972). Each lane contains 10 µg of polyadenylated RNA taken at the indicated time post deflagellation. The blot was probed with 0.7 kb of FA2 cDNA and re-probed with 0.5 kb of CBL cDNA and 0.5 kb of ß-tubulin cDNA as controls. (D) Ratio of FA2 message compared with that of CBL.

View larger version (49K): [in a new window]   Fig. 4. Localization of Fa1p in fa2 mutant cells. Western blot of flagellar-basal body complexes (FBBCs) isolated from wild-type, fa1-4 and fa2-4 strains. Thirty micrograms of protein were loaded into each lane and the blot was probed with a polyclonal antibody recognizing the C-terminus of FA1.

View larger version (51K): [in a new window]   Fig. 5. Cell size distribution of log phase cells. (A) Photomicrographs of wild-type (B214) and fa2-2 cells. The fa2-2 cells are larger (on average) with respect to length, width and volume. (B) Size distribution of wild-type, fa2-2 and fa2-2 rescued cells. Cell volumes were calculated from length and width measurements as described in Materials and Methods. The mean volumes were: wild type (151 µm3), fa2-2 (287 µm3) and fa2-2 rescued (152 µm3). One hundred cells from each culture were measured.

View larger version (17K): [in a new window]   Fig. 6. Cell size distribution of synchronized cells and multiple fission of asynchronous cells. (A) Wild type and fa2-2 mutant cells were grown in minimal media for several days in constant light, then placed in the dark for 24 hours. Cell sizes were measured immediately following exposure to light and again after 10 hours of continuous light. One hundred cells from each culture were measured. (B) Wild-type and fa2-2 mutant cells were grown in rich (TAP) media in constant light for several days. Aliquots of the culture were plated on minimal media and placed in the dark for 24 hours. Individual cells/colonies were scored for having undergone 0, 1, 2, 3 or 4 divisions during the 24 hour incubation in the dark by counting cells in micro-colonies. 300 cells were scored for each culture from each of two independent experiments.

View larger version (21K): [in a new window]   Fig. 7. Cell cycle analyses. (A-D) FACS analysis of synchronized wild-type and fa2-2 cultures. Cells were synchronized by incubation in the dark for 24 hours. Samples were taken at 0, 10 and 21 hours after return to the light and prepared for flow cytometric analysis. DNA content per cell, measured by fluorescence intensity of incorporated propidium iodide, is plotted in a histogram depicting relative cell numbers at each intensity. (E) Cultures were incubated in the dark for 24 hours and then returned to the light. Aliquots were then analyzed microscopically at the indicated time points for evidence of mitotic cleavage furrows. The percentage of cells with cleavage furrows was scored as the % of cells in M phase. Three hundred cells were scored for each culture.

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