, 2011). Depending on these criteria, we picked 6 candidate proteins and determined their subcellular localization by epitope tagging at their endogenous
locus. One particular candidate protein (Tb927.1.4310), which includes a predicted molecular mass of 183 kDa and
consists of four C-terminal coiled-coiled motifs, localized on the FAZ filament (Fig. 1A), as shown by co-
localization with FAZ1 and CC2D, two method identified FAZ filament proteins (Vaughan et al., 2008; Zhou et al.,
2011). We named this protein FAZ2 (Flagellum Attachment Zone protein 2) immediately after FAZ1, the 1st recognized
FAZ filament protein in T. brucei (Kohl et al., 1999; Vaughan et al., 2008). FAZ2 appeared for being
conserved in T. cruzi and Leishmania, but no homolog was discovered outside the kinetoplastids.
FAZ2 RNAi triggers flagellum detachment and defective cytokinesis
To investigate the perform of FAZ2, RNAi was carried out during the procyclic type. To monitor the
efficiency of RNAi, we tagged the endogenous FAZ2 using a triple HA epitope in cells harboring the
FAZ2 RNAi construct. Western blot showed that upon RNAi induction, FAZ2::3HA remained within the
cytoskeletal pellet fraction but was slowly decreased to an undetectable degree right after 48 h of RNAi (Fig.
1B). This depletion of FAZ2 inhibited cell proliferation (Fig. 1C), leading to the sellckchem accumulation of cells
with many (>2) nuclei to ~50% of the complete population after RNAi for 72 h (Fig. 1D), suggesting
defective cytokinesis. Quite possibly the most striking defect was flagellum detachment, which occurred as early as sixteen h
after RNAi, and right after 48 h far more than 80% of the cells contained detached flagella (Fig.
detachment occurred on all cell forms, together with 1N1K (1 nucleus and 1 kinetoplast), 1N2K, 2N2K
and polyploid cells.
FAZ2 RNAi disrupts the FAZ filament and destabilizes FAZ filament proteins
Flagellum detachment appeared to get attributed to defective assembly of your new FAZ filament as
proven by anti-FAZ1 immunostaining. In all FAZ2-depleted cells which has a detached flagellum, FAZ1 was
detected at the short, new FAZ filament and the full-length previous FAZ filament (Fig. 2A), suggesting that
FAZ2 is needed for assembly with the new FAZ, but not the outdated FAZ. As a consequence of mal-formation from the new
FAZ filament, we hypothesized Olaparib that FAZ1 was accumulated inside the cytosol on FAZ2 RNAi.
To test this
chance, we prepared cytosolic and cytoskeletal fractions for western blot to watch the distribution
and degree of FAZ1, which was endogenously tagged that has a triple HA epitope in FAZ2 RNAi cells. FAZ2
RNAi was induced for 24, 48 and 72 h, and western blot was carried out to detect FAZ1 degree in the
cytosolic and cytoskeletal fractions. The results showed that FAZ1 level during the cytoskeletal fraction
commenced to lower after RNAi for 24 h and was lowered to an incredibly very low degree soon after 72 h (Fig. 2B).