Morphological features of TbIC138 knockdown.

<p>DIC images showing examples of attached (A), partially detached (B) and fully detached (C) flagella. Scale bar represents 5 μm. D) Graph showing flagellar lengths and total cell lengths of uninduced, 48 hours- and 72 hours-post-induction IC138^<i>RNAi</i> cells. Error bars show standard deviation, number of samples (n) is indicated below each point.</p

Tagged TbIC138 protein: extent of knockdown and localization.

<p>A) Western blot showing detection of IC138::3xHA (98 kD) and Tubulin (50 kD) in representative examples of IC138^<i>RNAi</i> cultures 0, 48 hours- and 72 hours-post-induction. B) Quantification of Western blot showing extent of knockdown of IC138 protein levels. The level of protein in uninduced cells (uninduced) was set as 1.0, and the relative expression in 48 hours- or 72 hours-post-induction cells is indicated. Data represent averages from three independent sets of uninduced and induced cultures, with standard deviation indicated by error bars. C) Western blot showing detection of IC138::3xHA, tubulin or trypanin (54 kD) in different fractions of IC138^<i>RNAi</i>::3xHA cultures: WC = Whole cell, CY = cytoskeleton, S1,P1 = supernatant and pellet, respectively, from low salt extraction, S2, P2 = supernatant and pellet, respectively, from high salt extraction.</p

Occurrence of phenotypes over time following knockdown of TbIC138.

<p>A) Frequency of cells with normal, slow and abnormal motility or immotile. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139579#pone.0139579.s007" target="_blank">S2 Table</a> for motility classification. B) Frequency of cells with attached, partially detached or fully detached flagella. C) Frequency of detached flagella showing normal movement, aberrant movement or paralysis. Error bars represent standard deviation between two independent experiments, keys are at left of each graph.</p

Knockdown of Inner Arm Protein IC138 in <i>Trypanosoma brucei</i> Causes Defective Motility and Flagellar Detachment

<div><p>Motility in the protozoan parasite <i>Trypanosoma brucei</i> is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged <i>In situ</i>-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in <i>T</i>. <i>brucei</i>.</p></div

Growth and sedimentation analyses of TbIC138 knockdown.

<p>A) A representative cumulative growth curve of IC138^<i>RNAi</i> uninduced (dashed line) or tetracycline-induced (solid line). Cell titer = cells/mL. B) Sedimentation assay showing the motility of IC138^<i>RNAi</i> cells in uninduced (dashed line) or induced (solid line) by monitoring absorbance at 600nm for a 10-hour period starting at 48 hours-post-induction. Error bars indicate standard deviation between replicate vials.</p