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First published online 8 April 2003
doi: 10.1242/jcs.00438

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Kinesin II and regulated intraflagellar transport of Chlamydomonas aurora protein kinase

University of Texas Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, TX 75235-9039, USA

View larger version (32K): [in a new window]   Fig. 1. Distribution of 78 kDa and 80 kDa isoforms of CALK in wild-type, unactivated gametes. (A) Both isoforms of CALK are primarily localized in the cell bodies of unactivated wild-type gametes. Flagella (30 µg) and cell bodies (15 µg) isolated from unactivated wild-type gametes were analysed by SDS-PAGE and immunoblotting with anti-CALK antibody. (B) Absence of membrane-associated CALK in unactivated gametes. Unactivated wild-type gametes were separated into freely soluble, NP-40-soluble and NP-40-insoluble fractions and analysed by SDS-PAGE and immunoblotting with anti-CALK antibody. (C) Sucrose gradient analysis of CALK of unactivated gametes. NP-40 extracts of unactivated wild-type gametes were separated on 5-20% sucrose gradients and equal portions of each fraction were analysed by anti-CALK immunoblotting. (We used NP-40 to disrupt the gametes in this experiment for consistency with the experiments in Fig. 2.) All 18 fractions from the gradient were analysed by SDS-PAGE and immunoblotting but only the top 9, which contained all of the CALK detected on the gradient, are shown here.

View larger version (47K): [in a new window]   Fig. 2. Translocated 78 kDa flagellar CALK required detergent for solubilization and is present in a more rapidly sedimenting complex in wild-type gametes. (A) 78 kDa CALK translocates into flagella during gamete activation. Cell bodies and flagella isolated from wild-type unactivated mt– gametes and wild-type mt– gametes activated by incubation in dibutyryl cAMP were analysed by anti-CALK immunoblotting. (B) Unlike cell body CALK, most translocated 78 kDa flagellar CALK in activated gametes requires detergent to be soluble. The freely soluble, NP-40-soluble (i.e. sedimented at 170,000 g after mechanical disruption and subsequently rendered soluble by addition of NP-40) and NP-40-insoluble fractions of flagella and cell bodies from activated wild-type mt– gametes were analysed by anti-CALK immunoblotting. (C) Translocated 78 kDa flagellar CALK is part of a higher molecular mass complex. NP-40 extracts, which contained both freely soluble proteins and proteins that required NP-40 to be soluble, were prepared from flagella and cell bodies from activated mt+ and mt– gametes, and fractionated on 5-20% sucrose gradients; the fractions were analysed by anti-CALK immunoblotting.

View larger version (24K): [in a new window]   Fig. 3. Fla10 protein levels and the rate of zygote formation in wild-type and fla10 gametes. (A) Fla10 protein in the flagella of wild-type and fla10 unactivated gametes was determined by SDS-PAGE and immunoblotting with anti-Fla10N antibody (top). The lower panel shows the same blot probed with an anti-LC8 antibody as a loading control. (B) Wild-type and fla10 mt– gametes were mixed with mt+ gametes and, at the indicated times, the extent of zygote formation was assessed. The figure shows zygote formation as percentage of the maximum zygote formation for each sample (70% for the wild type and 42% for fla10). , wild-type; , fla10.

View larger version (32K): [in a new window]   Fig. 4. The 80 kDa isoform of CALK is mislocalized in fla10 gametes and is associated with the NP-40-insoluble, axonemal fraction of flagella. Wild-type and fla10 vegetative cells, and unactivated gametes (A) were analyzed by anti-CALK immunoblotting, as were flagella isolated from wild-type and fla10 vegetative cells and unactivated gametes (B). (C) Freely soluble, NP-40-soluble and NP-40-insoluble fractions of flagella and cell bodies isolated from unactivated fla10 gametes were analysed by anti-CALK immunoblotting.

View larger version (29K): [in a new window]   Fig. 5. Regulated translocation of 78 kDa CALK into flagella is disrupted in fla10 gametes. Wild-type and fla10 mt– gametes were activated by incubation in dibutyryl cAMP and flagella and cell bodies were isolated and analysed by anti-CALK immunoblotting.