CMF70 is a subunit of the dynein regulatory complex

Abstract

SummaryFlagellar motility drives propulsion of several important pathogens and is essential for human development and physiology. Motility of the eukaryotic flagellum requires coordinate regulation of thousands of dynein motors arrayed along the axoneme, but the proteins underlying dynein regulation are largely unknown. The dynein regulatory complex, DRC, is recognized as a focal point of axonemal dynein regulation, but only a single DRC subunit, trypanin/PF2, is currently known. The component of motile flagella 70 protein, CMF70, is broadly and uniquely conserved among organisms with motile flagella, suggesting a role in axonemal motility. Here we demonstrate that CMF70 is part of the DRC from Trypanosoma brucei. CMF70 is located along the flagellum, co-sediments with trypanin in sucrose gradients and co-immunoprecipitates with trypanin. RNAi knockdown of CMF70 causes motility defects in a wildtype background and suppresses flagellar paralysis in cells with central pair defects, thus meeting the functional definition of a DRC subunit. Trypanin and CMF70 are mutually conserved in at least five of six extant eukaryotic clades, indicating that the DRC was probably present in the last common eukaryotic ancestor. We have identified only the second known subunit of this ubiquitous dynein regulatory system, highlighting the utility of combined genomic and functional analyses for identifying novel subunits of axonemal sub-complexes.

Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

“…For ACP1 knockdowns, cells were grown with or without tetracycline at 1 g/ml for 72 h prior to harvesting. Total RNA was extracted using a Qiagen RNeasy kit, and quantitative reverse transcriptase, real-time PCR (qRT-PCR) was performed as described previously (45). Gene-specific primer sets were designed using the Trypanofan RNAit algorithm (46) and the NCBI Primer-BLAST program (47).…”

“…qRT-PCR was conducted on a DNA Engine Opticon 2 real-time cycler (MJ Research, Bio-Rad) using iQ SYBR green Supermix (Bio-Rad) according to the manufacturer's instructions. All analyses were performed in technical duplicate on at least two independent RNA preparations, and values were normalized against those for two stage-independent control genes, TERT and PFR2 (48), using the 2 Ϫ⌬⌬CT method (45,49). In situ epitope tagging.…”

“…Lysates were centrifuged for 20 min at 15,000 ϫ g at either 4°C or room temperature to separate solubilized proteins (S) from the insoluble pellet (P) fraction. Immunoblotting was done as previously described (45).…”

CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei

“…Procyclic 29-13 cells were subjected to detergent fractionation using a two-step fractionation method as previously described (25,38). Nonionic 1% Nonidet P-40 (NP-40) was used to solubilize the cell, and the pellet fraction was subsequently extracted in 0.5 M and 1 M NaCl PMN (10 mM NaPO 4 , pH 7.4, 1 mM MgCl 2 , 150 mM NaCl) buffer.…”

“…Several CMF genes encode proteins determined to have known or implicated functions in axoneme motility through independent studies. These include subunits of the nexin-dynein regulatory complex (NDRC; trypanin, CMF44, CMF46, CMF70) (22)(23)(24)(25)(26), protofilament ribbon proteins (CMF2, CMF3, CMF4, CMF19) (27)(28)(29), dynein subunits (CMF39, CMF73) (30,31), and the trypanosome orthologue of the move backwards only 2 (MBO2) gene product (CMF8) (32,33). However, a large number of CMF proteins have not yet been subjected to direct, in-depth analysis.…”