The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis

Abstract

Nucleotide-binding proteins Nubp1 and Nubp2 are MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins and are conserved, essential proteins throughout the Eukaryotes. They have been implicated, together with their interacting minus-end directed motor protein KIFC5A, in the regulation of centriole duplication in mammalian cells. Here we show that Nubp1 and Nubp2 are integral components of centrioles throughout the cell cycle, recruited independently of KIFC5A. We further demonstrate their localization at the basal body of the primary cilium in quiescent vertebrate cells or invertebrate sensory cilia, as well as in the motile cilia of mouse cells and in the flagella of Chlamydomonas. RNAi-mediated silencing of nubp-1 in C. elegans causes the formation of morphologically aberrant and additional cilia in sensory neurons. Correspondingly, downregulation of Nubp1 or Nubp2 in mouse quiescent NIH 3T3 cells markedly increases the number of ciliated cells, while knockdown of KIFC5A dramatically reduces ciliogenesis. Simultaneous double silencing of Nubp1 + KIFC5A restores the percentage of ciliated cells to control levels. We document the normal ciliary recruitment, during these silencing regimes, of basal body proteins critical for ciliogenesis, namely CP110, CEP290, cenexin, Chibby, AurA, Rab8, and BBS7. Interestingly, we uncover novel interactions of Nubp1 with several members of the CCT/TRiC molecular chaperone complex, which we find enriched at the basal body and recruited independently of the Nubps or KIFC5A. Our combined results for Nubp1, Nubp2, and KIFC5A and their striking effects on cilium formation suggest a central regulatory role for these proteins, likely involving CCT/TRiC chaperone activity, in ciliogenesis.

A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells

“…The bound antibody was eluted with 2 9 1 mL of 100 mM glycine containing 50 % v/v glycerol at pH 2.2; fraction 2 of the purified antibody was used in 1:100 dilution for IF and 1:250 for WB. Affinity purified rabbit antibodies to Nubp1 and Nubp2 [42] were used at 1:300 dilution for WB. Additional primary and secondary antibodies are listed in Table S2.…”

“…The four proteins were expressed in E. coli strain BL21(DE3)pLysS; KATNAL2-S1 and -L1 were produced as GST fusions (Table S1), after a 5-h induction with 0.5 mM IPTG (-S1) or a 3-h induction with 0.05 mM IPTG (-L1), or a 5-h induction with 0.5 mM IPTG (GST-only, negative control), all at 20°C. Nubp1 and Nubp2 were expressed as His-tagged fusions from pRSET A [42], under the same conditions as for KAT-NAL2-S1. Bacterial pellets (1.5 mL of liquid culture) were sonicated in 200 lL lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 1 % v/v Tween 20, 1 mM b-mercaptoethanol and 19 Complete protease inhibitor), centrifuged at 13,000 rpm for 15 min at 4°C and the soluble fraction, containing the recombinant protein retrieved (1/20 in each case was used as ''Input'' in Fig.…”

“…Immunofluorescence and live imaging IF was performed as described in [42]. Table S1) at the extremities of the predicted ORF of mouse Katnal2 XM_006526437.…”

“…We have recently shown that nucleotide-binding proteins Nubp1 and Nubp2 are core components of cilia and flagella in different species and RNAi knockdown of either Nubp demonstrated their role as key regulators of ciliogenesis [42]. Here, we conducted a yeast two-hybrid screen to identify protein interactions for Nubp1.…”

“…Additionally, because of the localization of KATNAL2s at the ciliary axoneme and basal body, their interactions with the Nubp proteins, shown to be regulators of ciliogenesis [42], and the known involvement of the archetypal katanin in axoneme dynamics in motile cilia [14,49], we also investigated the possible role of KATNAL2 proteins specifically in ciliogenesis.…”