Fig 1.
FAP265 localizes in flagella, basal bodies and cytoplasm in vegetative Chlamydomonas cells.
(A) Coomassie gel showing lysates from whole cell and flagella of CC-4533 cells, separated on 12% SDS-PAGE. Western blotting using antibodies generated against recombinant FAP265 detects a specific band at ~16.2 kDa both in whole cell and flagellar fractions. Further, the blots were also probed with antibodies against NAB1 (Cytosolic marker). (B) Vegetative cells of wild type CC-4533 stained with antibodies against α-tubulin (green) and FAP265 (red). Arrows mark flagella and arrow head marks basal bodies. Nuclei were stained with DAPI (cyan). Scale: 10μm.
Fig 2.
FAP265 localizes in the nucleus, (A, B) and at the cleavage furrow (C) in dividing CC-4533 cells.
CC-4533 cells at different stages of cell division were fixed in methanol and stained with antibodies against α-tubulin (green) and FAP265 (red). Arrow heads mark nucleus and arrows mark cleavage furrow. Nuclei were stained with DAPI (cyan). Scale: 10μm.
Fig 3.
Validation of fap265 mutant strain.
(A) PCR amplification using FAP265 gene specific primers showed a specific band at ~2.0 kb with CC-4533 but not with fap265 mutant genomic DNA. (B) Equal amount of whole cell lysates from CC-4533 and fap265 mutant cells were separated on 12% SDS-PAGE; western blotting using antibodies against recombinant FAP265 detects a specific band at ~16.2 kDa in CC-4533 but not in fap265 lysates. Western blotting using antibodies against α-tubulin was used as loading control. (C) CC-4533 and fap265 cells were stained with antibodies against FAP265 (Red) and Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI (cyan). Scale: 10μm.
Fig 4.
Loss of FAP265 results in severe hatching defects and failure of daughter cells to assemble flagella in sporangium.
(A) Bright field micrographs showing sporangium containing large number of daughter cells at steady state in fap265 mutant population compared with control cells. fap265 mutant cells are significantly bigger compared with wild type cells. Arrows mark flagella. Scale: 20μm (B) fap265 mutants and control cells in dividing stage were stained with antibodies against Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI. Scale: 10μm.