QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 April 2006
doi: 10.1242/jcs.02900

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pradel, L. C. Articles by Robinson, D. R. Search for Related Content PubMed PubMed Citation Articles by Pradel, L. C. Articles by Robinson, D. R. Social Bookmarking                         What's this?

NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei

Laboratoire de Génomique Fonctionnelle des Trypanosomatides, CNRS UMR 5162, Université Bordeaux 2, 146 rue Léo Saignat, Bât. 3A, 33076 Bordeaux CEDEX, France

View larger version (83K): [in a new window]   Fig. 1. (A) Schematic representation of TbNRKC, HsNEK1 and HsNEK2 proteins. TbNRKC contains a N-terminus kinase domain, a coiled-coil domain (CC), two PEST motifs, but does not contain a leucine zipper motif (LZ). HsNEK1 contains a N-terminus kinase domain, several coiled-coil domains and two PEST domains but no LZ motif, KEN or D-Box. HsNEK2 contains a coiled-coil domain, a leucine zipper, a KEN-box, a D-box and a phosphatase protein1 binding site (PP1). (B) The kinase domain of TbNRKC is highly conserved in NEKs. Residues conserved in more than 60% of the proteins are dark grey. Similar residues are light grey. Sequences used: TbNRKC (DQ054526), HsNek1 (Q96PY6), HsNek2 (P51955), DmNek2 (NM_132187), AnNIMA (P11837), DdNek2 (SLD805), PfNek1 (CAB76949), CrFa2 (AAL86904) and TbNrkA (Q08942).

View larger version (65K): [in a new window]   Fig. 2. (A) Purified recombinant TbNRKC-6His protein (NRKC, black left arrowhead) phosphorylates /ß casein substrate in vitro and preferentially phosphorylates ß casein (lane 2 arrowhead). The negative control BSA (apparent molecular mass 67 kDa) is not phosphorylated (lane 3). Purified kinase-dead TbNRKC-6His protein (mNRKC) (red arrowhead) does not phosphorylate the /ß casein (lane 7) or BSA (lane 8). Controls: lane 1 TbNRKC alone, lane 4 /ß casein alone, lane 5 BSA alone, lane 6 mNRKC alone. (B) Corresponding Coomassie-Blue-stained SDS-PAGE as protein loading control. (C) Corresponding western blot with Isa-1 polyclonal antibody as recombinant protein loading control. Pre-stained (left) and non-prestained (right) molecular mass markers show a slight discrepancy in the relative migration in the gel. Note that the recombinant mNRKC protein (B, position indicated by red arrowhead) migrates at a lower molecular mass than the recombinant NRKC (B, position indicated by black arrowhead). Arrowheads indicate where mNRKC and NRKC would normally run on SDS-PAGE.

View larger version (81K): [in a new window]   Fig. 3. (A) The polyclonal antibody Isa-1 recognises the 90 kDa TbNRKC protein, which is expressed in the procyclic (Pro) and bloodstream forms (BS) of T. brucei (2.5x106 cells/well loaded) on western blot. Controls: pre-immune on procyclic (lane 1) and bloodstream (lane 2) forms, no primary antibody (lanes 5 and 6). (B) The Isa-1 polyclonal antibody specificity to the NRKC protein was tested on western blot of the purified recombinant TbNRKC protein (lanes 1 and 2) and whole cells (lanes 3 and 4) without (-) or with (+) pre-incubation of Isa-1 with the recombinant protein (saturation assay). (C) TbNRKC is a basal body protein. T. brucei cytoskeletons (a-d) were prepared for immunofluorescence. A basal body and weak FAZ signals were observed using Isa-1 (c). Isa-1 specificity was assessed by saturation assays (e-h). A tetracycline induced (48 hours) NRKC-GFP expressing cell (i-l) illustrates direct GFP fluorescence on the basal bodies. Cytoskeletons of NRKC-GFP-expressing cells (m-p) probed with anti-GFP antibody (n) and YL-1/2 (o) confirms the basal body location of TbNRKC. a,e,i, phase-contrast images; m, phase-contrast image plus DAPI staining. Bar, 5 µm. (D) Flagella probed with Isa-1 visualised by electron microscopy. The majority of the Isa-1 labelling is on the proximal end of the mature basal body (arrowhead), some label is observed on the transition zone. The Isa-1 labelling of the immature basal body (arrow) cannot be clearly defined as proximal or distal because the immature basal body in vivo is orthogonal to the mature basal body. Some labelling can be observed between the mature and the immature basal bodies. Bar, 250 nm.

View larger version (44K): [in a new window]   Fig. 4. (A) Growth curve of TbNRKC RNAi-induced cells. Knockdown of TbNRKC is not lethal and cell growth is similar to non-induced cultures up to 10 days after induction. (B) Isa-1 immunofluorescence signal is present in non-induced cells (-TET) on the basal body (c). 48 hours after induction, Isa-1 signal is lost in RNAi-induced cells (+TET) (d). a,b, phase-contrast images. Bar, 5 µm. (C) TbNRKC levels analysed by western blot after RNAi induction (in days). 2 days of induction (+) is sufficient to deplete cells of TbNRKC protein. No reappearance of TbNRKC protein is detected up to 10 days of induction. The transformed cell line at time zero (0) or non-induced cells (-) express similar amounts of TbNRKC protein as wild-type cells (WT). Membranes were probed with Isa-1 (upper panel) or with L8C4 as a loading control (lower panel). 2.5x106 cells/well were loaded.

View larger version (54K): [in a new window]   Fig. 5. Phenotypes induced by RNAi in procyclic cells. (A) A small population of cells (1%) displayed flagella pairs. Cytoskeletons of cells induced for 48 hours probed with YL1/2 (b) and M25 plus DAPI (c). (B) A small population of cells displayed clusters of non-flagellated basal bodies. Cells were probed as in A. (C) A RNAi-induced four-basal-body cell, probed with YL1/2 (b) and M25 plus DAPI (c), illustrating a normal phenotype. Knockdown of TbNRKC does not block basal body separation or cytokinesis. Bars, 5 µm. (D) Basal body counts of wild-type (WT) and TbNRKC RNAi-induced cells (+TET). After 72 hours of induction, 46.2% of the population had two basal bodies (2BB) compared with 77.04% of wild-type cells. 47.8% of induced cells had four basal bodies (4BB) (two mature and two immature) compared with 22.2% in wild-type cells. 4.6% displayed more than four basal bodies (>4BB), 0.4% displayed clustered basal bodies (Clust. BB) and 1% displayed mature flagella pairs (MAT. PAIRS). Values are mean ± s.e.m. of three experiments. (E) FACS analysis of RNAi knockdown cells. After 72 hours (72 H) of RNAi induction, the percentage of cells in G1 decreased from 61.17% (WT cells) to 51.83%. The percentage of cells in S-phase remained stable (11.23% WT, 12.83% RNAi). The percentage of cells in G2-M phase increased (from 24.93% WT to 28.78% RNAi). The percentage of cells with abnormally high DNA content also increased (from 3.02% WT to 6.87% RNAi).

View larger version (40K): [in a new window]   Fig. 6. Overexpression of TY1-NRKC blocks cytokinesis. (A, top panel) Western blot of overexpressed TY1-NRKC protein (time in hours). The membrane was probed with Isa-1 and indicates the increased concentration of TY1-NRKC of protein observed in induced (+) cells. The blot was also probed with the anti-TY1 tag antibody BB2. Wild-type cells (WT), induced time 0 or non-induced (-) cells did not express the tagged protein. TY1-NRKC is observed after 24 hours of induction as illustrated in (+) lanes. The blot was probed with anti-PFRA monoclonal L8C4 as a loading control. Blots were loaded with 5x106 cells/lane. (A, bottom panel) Comparative growth curves of non-induced (- TET) and induced cells (+ TET) indicate that cells overexpressing TY1-NRKC have a reduced growth rate compared with non-induced cells. (B) Cytoskeletons of TY1-NRKC cells induced for 48 hours were probed by immunofluorescence with YL1/2 (b) and M25 (c). This multinucleated cell possesses one flagellum but four basal bodies (b and d), indicating that the cell passed through the cell cycle the absence of cytokinesis. The arrowheads in c and d indicate a large kinetoplast close to the basal bodies. Bar, 5 µm. (C) Counts of cells possessing at least one large kinetoplast and more than two nuclei in non-induced (- TET) and induced cells (+ TET) from 0 to 96 hours. After 96 hours of induction, 80.8% of cells possessed at least one large kinetoplast and more than two nuclei. (D) Populations of non-induced (- TET) and induced (+ TET) cultures were assessed to identify the percentage of cells possessing two basal bodies (2 BB), four basal bodies (4 BB), more than four basal bodies (>4 BB) and clusters of non-flagellated basal bodies (Clust. BB). Assessment was done from 0 to 96 hours of TY1-NRKC induction. All values are mean ± s.e.m. of three experiments.

View larger version (102K): [in a new window]   Fig. 7. (A) Longitudinal section of a TY1-NRKC non-induced cell illustrating the mature flagellum (arrow), its mature basal body (upper black arrowhead) and its immature pro-basal body (lower black arrowhead). This cell is in the early stages of kinetoplast replication and possesses a second immature pro-basal body (lower white arrowhead), which is linked to a second flagellum (not within the plane of this section). (B) Longitudinal section of a TY1-NRKC-induced cell (72 hours). The section traverses a cluster of seven basal bodies (arrowheads). At least three non-flagellated basal bodies can be observed. The kinetoplast is larger than wild-type cells (at least 3 µm in diameter). K, kinetoplast; FP, flagellar pocket. Asterisks indicate ribosome-free regions of the TAC. Bars, 500 nm.

View larger version (53K): [in a new window]   Fig. 8. Overexpression of kinase-dead TbNRKC in vivo. (A, top panel) Western blot of wild-type (WT), non-induced (-) and induced cells (+) for 72 hours. The membrane was probed with Isa-1 and indicates the overexpression of mNRKC in induced cells. (A, bottom panel) Growth curve of non induced (-) and induced cells (+). (B) Basal body counts of non-induced (- TET) and induced cells (+TET). After 72 hours of induction, 48.57% of induced cells had two basal bodies (2BB) compared with 63.28% of non-induced cells. 47.04% of induced cells had four basal bodies (4BB) (two mature and two immature) compared with 35.78% of non-induced cells. In induced cells 3.63% (0% for non induced) displayed more than four basal bodies (>4BB), 0.19% (0% for non induced) displayed clustered basal bodies (Clust. BB) and 0.57% displayed mature flagella pairs (MAT. PAIRS). (C) A cell overexpressing kinase-dead TbNRKC in vivo probed with YL1/2 (b) and M25 plus DAPI (c), illustrating the four-basal-body phenotype. Bar, 5 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter