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First published online 19 July 2005
doi: 10.1242/jcs.02455

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A NIMA-related kinase, Cnk2p, regulates both flagellar length and cell size in Chlamydomonas

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada

View larger version (22K): [in a new window]   Fig. 1. Cnk2p-HA is an axonemal protein. (A) Schematic representation of the CNK2-HA expression construct, containing the CNK2 coding sequence (CDS) with the 3XHA epitope tag on the C-terminus. The PsaD promoter and UTRs are used to drive constitutive expression (Fischer and Rochaix, 2001). E, EcoRI; H, HindIII; N, NdeI restriction sites. (B) Immunoblot analysis of cell fractions from Cnk2p-HA expressing cells probed with an anti-HA antibody. Blots were stripped and reprobed with an anti--tubulin antibody. Cell equivalent amounts of whole cells (WC), cell bodies (CB) and flagella (Flag) were loaded in each lane (left-hand blot). Stoichiometric amounts of flagella (Flag), membrane and matrix fraction (M+M), and axonemal fractions (Axo) were loaded in each lane (right-hand blot). (C) Wild type and two examples of Cnk2p-HA cells triple-stained for indirect immunofluorescence with anti--tubulin, anti-HA and anti-centrin antibodies. Bar, 5 µm.

View larger version (16K): [in a new window]   Fig. 2. Cnk2p-HA is not associated with established axonemal accessory structures. Indirect immunofluorescence of cells expressing Cnk2p-HA in axoneme structural mutant backgrounds using anti-HA and anti--tubulin. pf3, lacks dynein regulatory complex; pf9,lacks inner dynein arms; pf14, lacks radial spokes; pf18, lacks central pair; oda9, lacks outer dynein arms. Bar, 5 µm.

View larger version (59K): [in a new window]   Fig. 3. Knockdown of CNK2 expression via RNAi. (A) Schematic representation of the CNK2-RNAi expression construct. The construct, designed to generate 834 bp of dsRNA upon expression, was used to produce stable RNAi strains by nuclear transformation. Bs, BsiWI; E, EcoRI; H, HindIII; N, NdeI; P, PstI restriction sites. (B) Northern analysis of CNK2 transcript levels in five independent CNK2-RNAi transformants. 15 µg total RNA was loaded in each lane. The blot was probed with 0.5 kb of 32P labeled CNK2 cDNA, stripped and reprobed with 0.5 kb of 32P labeled ß-tubulin cDNA. (C) The ratio of CNK2 message to tubulin message was determined by comparing band intensities using ImageQuant v2.0. CNK2-RNAi strain 20 was used in all subsequent experiments.

View larger version (22K): [in a new window]   Fig. 4. Aberrant Cnk2p levels affect cell size and flagellar length distributions. (A) Flagellar length distributions for the wild type (WT), Cnk2p-HA and CNK2-RNAi. (B) Cell volume distributions for wild type, Cnk2p-HA and CNK2-RNAi. For each strain, 100 cells or flagella (one per cell) were measured in three independent experiments and pooled to generate the distributions.

View larger version (26K): [in a new window]   Fig. 5. Analysis of flagellar dynamics in Cnk2p-HA and RNAi strains. (A) Flagellar lengths were measured during recovery from pH shock in the presence or absence of 10 µg/ml cycloheximide. 40 flagella were measured for each time-point for each of three independent experiments, error bars represent s.e.m. (B) Flagellar lengths during resorption of flagella in fla10-1:Cnk2p-HA and fla10-1:CNK2-RNAi strains after shift to restrictive temperature. 40 flagella were measured for each time-point for each of three independent experiments, error bars represent s.e.m. (C) Cnk2p-HA cells assembling flagella post deflagellation and disassembling flagella prior to mitosis, triple-stained for indirect immunofluorescence with anti--tubulin, anti-HA and anti-centrin. Bar, 5 µm.

View larger version (24K): [in a new window]   Fig. 6. Aberrant Cnk2p levels affect cell cycle progression. (A) Mitotic index measurements of wild-type (WT), CNK2-HA, CNK2-RNAi and fa2-1 cultures. Cultures were synchronized by alternating light-dark cycles for 3 days and examined microscopically on day 4 for the percentage of cells with cleavage furrows. A total of 200 cells were examined for each time-point, from two independent experiments (100/experiment). (B) Growth of WT, CNK2-HA and CNK2-RNAi cells. Cultures were grown in minimal media for several days in constant light and placed in the dark for 24 hours. Cultures were shifted back to the light to initiate growth and samples were taken every 2 hours. A total of 200 cells were measured for each time-point from two independent experiments (100/experiment), error bars represent s.e.m. (C) Analysis of the number of division cycles post commitment. Cultures were grown in TAP media for several days, plated on minimal media, and placed in the dark for 24 hours. Colonies were examined microscopically to determine the number of cells per colony. A total of 600 colonies were scored from two independent experiments (300/experiment). (D) Commitment size measurement. Cultures were grown as in B, and a sample was fixed immediately after the shift to light. A total of 300 cells were measured from each culture in three independent experiments (100/experiment).