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First published online 11 July 2006
doi: 10.1242/jcs.03028

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A kinesin-like calmodulin-binding protein in Chlamydomonas: evidence for a role in cell division and flagellar functions

Dartmouth College, Department of Biological Sciences, 301 Gilman, Hanover, NH 03755, USA

View larger version (31K): [in a new window]   Fig. 1. (A) Diagram of domains within complete coding sequence of CrKCBP. CrKCBP contains a myosin tail homology domain (MyTH4), a band 4.1 domain (B41), present in a number of proteins that interface between the plasma membrane and cytoskeleton, a coiled-coil neck region (CC), a kinesin motor domain (KMD), and a calmodulin-binding domain (CBD). (B) RNA blot. 10 µg of poly(A)+ selected RNA prepared from control cells (lane 1), 45 minutes after deflagellation (lane 2) or 45 minutes after induction of flagella resorption (lane 3) were loaded into each lane. The blot was probed with a DNA fragment at the 5' end of the CrKCBP coding sequence (labeled Probe in A) as well as the S14 gene encoding ribosomal S14 protein to serve as a loading control. The band of approximately 1 kb represents the S14 transcript. The band at approximately 4.4 kb represents the CrKCBP transcript. This transcript increases in abundance following deflagellation and decreases slightly in abundance during flagellar resorption.

View larger version (16K): [in a new window]   Fig. 2. Immunoblot. Flagella (Fl) were isolated from wild-type cells and demembranated as described in Materials and Methods. The resulting membrane and matrix fraction (M) and axonemes (Ax) were isolated by centrifugation. Axonemes were divided and extracted with either 0.6 M NaCl (NaCl), 1 mM ATP (1ATP), or 5 mM ATP (5ATP) to produce extracted axonemes (EA) and axonemal extracts (E). The resulting extracted axonemes and extracts were separated by centrifugation and extracted axonemes were resuspended in an equal volume of buffer. For immunoblot analysis, 25 µl of each sample were loaded in each lane and anti-CrKCBP antibodies were used as a probe. These antibodies recognize a single band of approximately 140 kDa. This protein is extracted from axonemes under conditions of high salt and ATP. CrKCBP appears to be of higher molecular mass in flagella because of the large quantity of flagellar membrane in this sample, which interferes with protein migration.

View larger version (104K): [in a new window]   Fig. 3. Localization of CrKCBP by indirect immunofluorescence. Axonemes were isolated as described in the Materials and Methods. Samples in the right column were extracted with 5 mM ATP prior to processing for immunofluorescence. The axonemes were double labeled for tubulin (top row, Texas Red secondary antibody), and CrKCBP (middle row, Alexa Fluor 488 secondary antibody). The merged images are shown in the bottom row where white indicates co-localization of tubulin with CrKCBP. CrKCBP localizes along the length of the axoneme and is extracted upon exposure to 5 mM ATP.

View larger version (54K): [in a new window]   Fig. 4. (A-C) Immunoblots. (A) Blots from microtubule-binding studies probed with the anti-CrKCBP antibody. Extracts (Ex) were prepared by extracting axonemes with 1 mM ATP and treating with apyrase. The resulting extracts were then added to microtubules (T) assembled in vitro or to an equivalent volume of buffer (B). The microtubules were sedimented, the supernatant collected (S) and the pellet (P) resuspended in an equivalent volume of buffer. In some experiments ATP was added to the resulting pellet (P+ATP) to assess ATP-sensitive binding of CrKCBP to microtubules. (Top panel) Microtubule binding was conducted in the absence of calmodulin under low Ca2+ conditions; (second panel) binding was performed in the presence of high Ca2+-calmodulin (+Ca2++CaM) using an extract prepared in low Ca2+ conditions (-Ca2+); (third panel) binding was performed in the presence of Ca2++CaM using an extract prepared in high Ca2+ conditions (+Ca2+). Microtubule binding of CrKCBP is only mildly sensitive to the presence of Ca2+-CaM. (B) Blots from immunoprecipitation experiments using anti-calmodulin antibodies for precipitation and probed with either anti-calmodulin (CaM) or CrKCBP antibodies. Axonemes were extracted with 1 mM ATP in high Ca2+ conditions; half of the resulting extract (Ex) was treated with apyrase (+apyrase) and half was not (-apyrase). Precipitations were carried out as described in Materials and Methods and the resulting unbound (un) and precipitated (IP) proteins were processed for gel electrophoresis. The antibodies precipitate virtually all of the calmodulin in the extract but do not precipitate CrKCBP. (C) Blots of sucrose gradient fractions probed with anti-CrKCBP antibodies (fractions 1-23=20-5% sucrose, respectively). CrKCBP sediments at approximately 11S, peaking in fraction 11.

View larger version (41K): [in a new window]   Fig. 5. Indirect immunofluorescence localization of CrKCBP during interphase. (A) Localization of tubulin (Texas Red secondary antibody) and CrKCBP (Alexa Fluor 488 secondary antibody) in Chlamydomonas interphase cells. Areas of co-localization appear white in the merged image. Lower panels are enlargements of the region with the greatest CrKCBP staining. The significant fraction of CrKCBP localizes near the base of the flagellar microtubules. Bars, top row, 10 µm; bottom row, 1 µm. (B) Immunoblot. Flagellar basal body complexes (FBB) and flagella (FL) were prepared from equal numbers of cells and equal amounts were loaded onto the gel. The resulting blot was probed with antibodies against CrKCBP as well as PF20, a known axonemal component.

View larger version (80K): [in a new window]   Fig. 6. Cellular localization of CrKCBP and centrin by indirect immunofluorescence. Interphase cells were double labeled with anti-centrin (Texas Red secondary antibodies) and CrKCBP (Alexa Fluor 488 secondary antibodies) antibodies. Regions of co-localization appear yellow in the merged images. Panels to the right of each main image show an end-on view of protein localization. Lower panels are enlargements of the regions with greatest CrKCBP staining. CrKCBP co-localizes with centrin in a very small region, most probably representing the distal connecting fiber. Bars, 1 µm.

View larger version (87K): [in a new window]   Fig. 7. Cellular localization of CrKCBP during mitosis as shown by indirect immunofluorescence. Cells were triple labeled with anti-centrin (Alexa Fluor 594 secondary antibodies), anti-CrKCBP (Alexa Fluor 488 secondary antibodies) and anti-tubulin (Alexa Fluor 350 secondary antibodies) antibodies. As the cells progress through mitosis the CrKCBP staining appears concentrated at the duplicated microtubule organizing centers. However, as the cells progress to cytokinesis CrKCBP appears to localize to the phycoplast. Bar, top row, 10 µm; bottom row, 1 µm.