Biochemical purification of flagellar Hsp70. Chlamydomonas flagellar matrix extracts (lanes 1) were incubated with ATP-agarose beads, and the depleted extract (lanes 2) was separated from bound material (lanes 3) by centrifugation; lanes 4 contain a 10-fold-heavier loading of the contents of lanes 3. Panel A shows a silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel loaded with equivalent fractions. Panel B shows an immunoblot of a gel identical to that used in panel A, probed with the monoclonal antibody MA3-006 originally used to identify the flagellar chaperone (1).

HSP70A is present in both cell bodies and flagella. Immunofluorescent localizations of (A and B) HSP70A in wild-type cells using affinity-purified anti-HSP70A carboxy-terminal peptide rabbit polyclonal antibodies and (C and D) epitope-tagged HSP70A in transformant cells using the anti-HA epitope mouse monoclonal antibody 12CA5 are shown. Both stained the cytoplasm of cell bodies brightly; a discontinuous, punctate signal was also seen along the lengths of the pair of flagella extending from each cell body. (E) Localizations with the antitubulin monoclonal antibody TUB 1A2 (Sigma, St. Louis, MO) showed smooth labeling along flagella, and (F) control experiments from which primary antibodies were omitted showed essentially no labeling. Bar = 10 μm.

RT-PCR analysis of HSP70A upregulation during flagellar assembly. Total RNA samples isolated from cells bearing full-length flagella (not deflagellated [NDF]) and cells regenerating flagella 30 min after pH shock deflagellation (DF) were subjected to RT-PCR using primers specific for mRNAs encoding (A) alpha tubulin, (B) beta tubulin, (C) cytoplasmic HSP70A, (D) the ribosomal protein S14, (E) a G protein beta subunit-like protein, or (F) chloroplast HSP70B. RT-PCR products were examined at five-cycle intervals, and examples were chosen to accurately compare the kinetics of product formation and, thus, initial mRNA abundances.

HSP70A is an abundant cytoplasmic protein also present in flagella. Immunoblot analysis of TP (10⁶ cells) and total FL (10 μg) from wild-type cells (WT) and a transformant strain expressing HSP70A::HA (HA) is shown. Panel A is a Coomassie blue-stained gel showing total protein loading. The remaining panels are immunoblots of replicate gels probed with (B) affinity-purified anti-HSP70A carboxy-terminal peptide polyclonal antibodies, (C) the anti-HA epitope monoclonal antibody 12CA5, (D) the broadly cross-reactive pan-Hsp70 monoclonal antibody MA3-006, and (E) an anti-HSP70B carboxy-terminal peptide antiserum (Research Genetics, Huntsville, AL).

Immunofluorescent colocalizations of HSP70A and the kinesin-II motor FLA10. Panel A is an immunoblot of TP (10⁶ cell equivalents) and total FL (10 μg) probed with the antikinesin-II monoclonal antibody K2.4 that recognizes the 90-kDa Chlamydomonas IFT kinesin-II Fla10 (4, 12, 16). Panels B and C show cells double labeled with affinity-purified anti-HSP70A carboxy-terminal peptide rabbit polyclonal antibodies (red channel) and the anti-FLA10 mouse monoclonal antibody K2.4 (green channel). Green and red signal overlap is perceived as yellow (the exact hue determined by the green/red ratio). Panels D and E show enlarged views of the boxed areas from panels B and C in which the red, green, and red-green overlay channels are separated for clarity. In addition, colocalization analysis (performed using Image J [http://rsb.info.nih.gov/ij/] and the RG2B plug-in, in which a saturated blue pixel is generated wherever nonzero red and green pixel values coincide) is shown as a measure of coincidence not dependent upon stoichiometry. Bar = 10 μm (B and C) and 20 μm (D and E).