Short Report 857 Trichoplein controls microtubule anchoring at the centrosome by binding to Odf2 and ninein Miho Ibi1,*, Peng Zou1,*, Akihito Inoko1,*, Takashi Shiromizu1, Makoto Matsuyama1,2, Yuko Hayashi1, Masato Enomoto1,3, Daisuke Mori4, Shinji Hirotsune4, Tohru Kiyono5, Sachiko Tsukita6, Hidemasa Goto1,3 and Masaki Inagaki1,3,‡ 1Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681, Japan 2The Foundation for Promotion of Cancer Research, Chuo-ku, Tokyo 104-0045, Japan 3Department of Cellular Oncology, Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya 466-8550, Japan 4Department of Genetic Disease Research, Osaka City University Graduate School of Medicine, Osaka 545-8586, Japan 5Virology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan 6Laboratory of Biological Science, Graduate School of Frontier Biosciences/Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan *These authors contributed equally to this work ‡Author for correspondence ([email protected]) Accepted 27 October 2010 Journal of Cell Science 124, 857-864 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.075705 Summary The keratin cytoskeleton performs several functions in epithelial cells and provides regulated interaction sites for scaffold proteins, including trichoplein. Previously, we found that trichoplein was localized on keratin intermediate filaments and desmosomes in well- differentiated, non-dividing epithelia. Here, we report that trichoplein is widely expressed and has a major function in the correct localization of the centrosomal protein ninein in epithelial and non-epithelial cells. Immunocytochemical analysis also revealed that this protein is concentrated at the subdistal to medial zone of both mother and daughter centrioles. Trichoplein binds the centrosomal proteins Odf2 and ninein, which are localized at the distal to subdistal ends of the mother centriole. Trichoplein depletion abolished the recruitment of ninein, but not Odf2, specifically at the subdistal end. However, Odf2 depletion inhibited the recruitment of trichoplein to a mother centriole, whereas ninein depletion did not. In addition, the depletion of each molecule impaired MT anchoring at the centrosome. These results suggest that trichoplein has a crucial role in MT-anchoring activity at the centrosome in proliferating cells, probably through its complex formation with Odf2 and ninein. Key words: Trichoplein, Microtubule (MT), Centriole, Odf2, Ninein, Cell proliferation Journal of Cell Science Introduction is duplicated in the preceding cell cycle, lacks this structure. The Intermediate filaments (IFs), together with microtubules (MTs) mother centriole directly docks MTs at its appendages, whereas the and actin filaments, form the cytoskeletal framework in the daughter centriole does not (Bornens, 2002; Bettencourt-Dias and cytoplasm of various eukaryotic cells. Unlike MTs and actin Glover, 2007; Bornens, 2008; Nigg and Raff, 2009). filaments, the protein components of IFs vary in a cell-, tissue- and The dynamics of IF network is well correlated with that of other differentiation-dependent manner. Type I and type II keratin proteins cytoskeletal networks including centrosome and MTs. IF-associated form heterodimers in epithelial cells, and their composition also proteins (IFAPs) are emerging as important regulators that depends on tissue type and the stage of differentiation. Two coordinate the remodeling and function of such cytoskeletal heterodimers associate with one tetramer, which assembles into networks. Members of the plakin family (such as plectin) were higher-order oligomers and forms 10 nm filaments. Keratin IFs identified as crosslinkers between IFs and other cytoskeletal associate with plasma membrane specializations such as elements including actin filaments and MTs. Although several desmosomes and hemidesmosomes in the periphery. Similarly to IFAPs have been identified as regulators of IF remodeling and other cytoskeletal IFs, keratin IFs also extend into the cytoplasm function, the function of IFAPs in other cytoskeletal systems is where they provide a scaffold for other cytoskeletal elements largely unknown (Fuchs and Cleveland, 1998; Houseweart and including centrosome and MTs (Eriksson et al., 1992; Steinert, Cleveland, 1998; Izawa and Inagaki, 2006; Godsel et al., 2008). 1993; Fuchs and Weber, 1994; Fuchs and Cleveland, 1998; Magin Nishizawa and colleagues (Nishizawa et al., 2005) previously et al., 2007; Pekny and Lane, 2007; Godsel et al., 2008; Eriksson reported that trichoplein keratin filament-binding protein et al., 2009). (trichoplein or TCHP) serves as a keratin IF scaffold protein. The centrosome is the primary MT-organizing center in animal Similarly to another keratin IF scaffold protein, Albatross cells. It is composed of two orthogonal centrioles surrounded by (Sugimoto et al., 2008), this protein has a trichohyalin and plectin pericentriolar material from which MTs emanate and elongate. homology domain (TPHD; also see supplementary material Fig. These paired centrioles are structurally distinct, reflecting their S3A), which has similarities to classical keratin IF scaffold different ages. The older one (a mother centriole) functions as a proteins (Izawa and Inagaki, 2006). In villous epithelial (non- template in the preceding cell cycle and carries distal to subdistal dividing) cells of the small intestine, trichoplein mainly appendages, whereas the younger one (a daughter centriole), which colocalizes with keratin IFs and the desmosome (Nishizawa et 858 Journal of Cell Science 124 (6) al., 2005). Here, we report that trichoplein is concentrated at the Results and Discussion subdistal to medial zone of both mother and daughter centrioles We first produced a rabbit polyclonal antibody against full-length in dividing epithelial and non-epithelial cells. RNA interference (FL) mouse trichoplein. In total cell lysates of EpH4, MTD1A (RNAi)-mediated trichoplein depletion inhibited MT anchoring (mouse mammary epithelial) or NIH3T3 (mouse fibroblastic), anti- at the centrosome. This MT-anchoring activity was regulated by mouse trichoplein reacted specifically with a band corresponding the interaction of trichoplein with two other centriole proteins, to mouse trichoplein, which shared 77% homology with human Odf2 and ninein. trichoplein (Fig. 1A). Treatment with mouse trichoplein-specific Journal of Cell Science Fig. 1. Trichoplein is localized at the centrosome in proliferating cells. (A)Characterization of an anti-human or mouse trichoplein antibody by immunoblotting of indicated cell lysates. In lower panels, EpH4 cells were treated with control (Cont.) or mouse trichoplein-specific siRNA (Sq.5 or Sq.6) for 48 hours. (B,C)Localization of trichoplein (Tricho.; green) and centrin-2 (Cen2; red) in mouse small intestine. Nuclei were also stained with DAPI (D; blue) in these and 2+ 2+ subsequent panels. (D)Ca -switch experiments. MTD1A cells were incubated in low-Ca medium (DMEM with 0.05 mM CaCl2) for 17–18 hours and then switched to normal growth medium containing >1.8 mM Ca2+ to induce cell junction assembly. Treated cells were subjected to immunocytochemistry with anti- mouse trichoplein and anti-desmoplakin (DP1/2) at the indicated time after switching. NCM indicates cells incubated in normal Ca2+ medium containing 10% FBS without the switch. (E–G) Trichoplein localization in EpH4 (E,F), HeLa, KE37, U251, or IMR90 (G) cells. Red indicates staining with anti--tubulin (E) or anti- centrin-2 (F,G). Boxes in main image indicate centrosome structures shown at high magnification (E,G). Scale bars: 10mm (B,D,E), 1mm (C,F), 5mm (G). Centriolar function of trichoplein 859 small interfering RNAs (siRNAs) reduced immunoreactivity to trichoplein appeared to have translocated to the cell–cell contact this band (Fig. 1A), confirming the specificity of this antibody. We area before desmosome formation. Trichoplein colocalized at the next stained mouse small intestine with the anti-mouse trichoplein desmosome with desmoplakin at 8 hours after the switch (Fig. antibody. In absorptive epithelial cells, the antibody signals were 1D). primarily observed at the cell–cell border where desmoplakin was In addition to keratin IFs and desmosome, trichoplein appeared localized (supplementary material Fig. S1A,B). Especially in the to localize to anti-centrin-2-positive punctate structures (which villi, where cells are well differentiated, the signals were also implies that they are centrioles) (Paoletti et al., 1996; Laoukili et detected as bundles at the apical region (Fig. 1B,C). These staining al., 2000) in the crypt where dividing cells exist (Fig. 1B,C). Such patterns confirm the previous observation that trichoplein localizes a phenomenon was less observed in the villi where cells cannot to keratin IFs and desmosomes in the epithelia (Nishizawa et al., divide (Fig. 1B,C). In EpH4 mouse mammary epithelial cells, anti- 2005). mouse trichoplein signals overlapped with anti--tubulin-positive To elucidate the behavior of trichoplein during the formation of spots (which implied centrosomes), although they were also cell–cell attachment, we performed a Ca2+-switch experiment (Fig. prominent at the cell-cell contact in cells with higher confluency 1D). In MTD1A cells, trichoplein was concentrated at the cell–cell (Fig. 1E). As shown in Fig. 1F, the number of anti-mouse trichoplein contact during cultivation in normal Ca2+ medium (NCM); signals