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Endogenous Localizome Identifies 43 Mitotic Kinesins in a Plant Cell

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Endogenous Localizome Identifies 43 Mitotic Kinesins in a Plant Cell Endogenous localizome identifies 43 mitotic kinesins PNAS PLUS in a plant cell Tomohiro Mikia,b, Haruko Naitoa, Momoko Nishinaa, and Gohta Goshimaa,b,1 aDivision of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan; and bMarine Biological Laboratory, Woods Hole, MA 02543 Edited by Ronald D. Vale, Howard Hughes Medical Institute and University of California, San Francisco, CA, and approved February 11, 2014 (received for review June 14, 2013) Kinesins are microtubule (MT)-based motor proteins that have available (12). Critically, the rate of homologous recombination been identified in every eukaryotic species. Intriguingly, land is unusually high in P. patens, such that gene disruption and plants have more than 60 kinesins in their genomes, many more epitope tagging are easy to perform. GFP or other fluorescent than that in yeasts or animals. However, many of these have not proteins can be fused to the endogenous protein, so that the yet been characterized, and their cellular functions are unknown. tagged proteins are expressed under the native promoter at the Here, by using endogenous tagging, we comprehensively deter- native chromosomal locus. P. patens spends most of its life cycle mined the localization of 72 kinesins during mitosis in the moss in a haploid state; therefore, the tagged protein substitutes the Physcomitrella patens. We found that 43 kinesins are localized to untagged protein (13, 15). The GFP-based localization deter- mitotic structures such as kinetochores, spindle MTs, or phragmo- mined in this way would thus likely represent the endogenous plasts, which are MT-based structures formed during cytokinesis. protein localization. Another key advantage of the P. patens Surprisingly, only one of them showed an identical localization system is the ease of live cell microscopy in caulonemal stem cells pattern to the animal homolog, and many were enriched at un- that actively divide every 5–6 h (13, 15). Caulonemal cells are expected sites. RNA interference and live-cell microscopy revealed found in protonemata, which are filamentous structures that ap- postanaphase roles for kinesin-5 in spindle/phragmoplast organi- pear after spore germination, and experience less autofluorescence- zation, chromosome segregation, and cytokinesis, which have not related interference derived from chloroplasts; therefore, they been observed in animals. Our study thus provides a list of MT- are suitable for the live imaging of GFP- or red fluorescent based motor proteins associated with the cell division machinery protein (RFP)-tagged markers during cell division (14, 19, 20). in plants. Furthermore, our data challenge the current generaliza- Previous studies have characterized the process of mitotic spin- tion of determining mitotic kinesin function based solely on stud- ies using yeast and animal cells. dle and phragmoplast formation in caulonemal cells by using GFP-tubulin, MAP65-GFP (anti-parallel MT-bundling protein), or EB1-GFP (MT plus-end-tracking protein) (14, 19, 22). Cau- ammalian kinesins have been classified into 14 subfamilies – lonemal cells, like many other plant cell types, do not have Maccording to sequence similarities (kinesin-1 kinesin-14) centrosomes or other discrete MT organizing centers; instead, (1, 2). A subset of kinesins plays critical roles during cell division, they generate MTs predominantly through amplification medi- where spindle bipolarity establishment, chromosome alignment ated by a protein complex called augmin (14, 23). Moreover, to the metaphase plate, spindle microtubule (MT) sizing, and unlike most other cell types in land plants, caulonemal cells do cytokinesis are ensured by the coordinated actions of kinesins, not form the preprophase band (PPB), a MT-based structure including the kinesin-4, -5, -6, -7, -8, -10, -12, -13, and -14 sub- that transiently appears at the early stages of mitosis in the cell families. These mitotic kinesins have been identified through cortex and determines the future division plane (24–26). Thus, functional analyses and localization studies in several organ- mitotic spindle/phragmoplast assembly in caulonemal cells appears isms. For example, GFP-tagging revealed four of six yeast to be a highly self-organized process that involves multiple force- kinesins localized to the kinetochores (3). Comprehensive generating factors; these features are also typically observed in RNA interference (RNAi) showed that 8 of 25 kinesins play acentrosomal spindles in animals (8, 27). a role in mitosis in the Drosophila S2 cell line (4). In humans, ∼20 of 45 kinesins have been identified as mitotic kinesins by RNAi and localization analyses using several cell types (2, 5, Significance 6). Most kinesins exert forces toward MTs or chromosomes; therefore, the identification of kinesins localized to the mi- It has been a mystery as to why plants have many more > totic apparatus has significantly contributed to the under- kinesins than do animals (e.g., 45 in humans, 60 in land standing of the mechanisms underlying mitosis, such as spindle plants). To address this issue, the experimental analysis of each bipolarity establishment, spindle elongation, or chromosome move- kinesin is critical. However, it has not been trivial to analyze > ment (7–9). 60 kinesins in a comprehensive manner in plants. Here, by Interestingly, recent whole genome sequencing has revealed using endogenous YFP tagging, we found that as many as 43 kinesins are localized to the mitotic apparatus in the moss that land plants possess many more kinesins in their genomes than Physcomitrella patens do animals. Arabidopsis thaliana has 61 kinesins, some of which . All but one kinesin showed a localiza- tion pattern distinct from the animal homologues, indicating cannot be assigned to the conventional 14 subfamilies (2, 10, 11). the necessity of studying individual kinesin functions in plants. Why they have so many kinesins is an open question, and this question cannot be addressed without experimentation. However, Author contributions: T.M. conceived the project; T.M. and G.G. designed research; T.M., it has been challenging to analyze more than 60 kinesins in a com- H.N., and M.N. performed research; T.M., H.N., M.N., and G.G. analyzed data; and T.M. prehensive manner in plants. To date, we have limited knowledge and G.G. wrote the paper. on the functions of the individual plant kinesins or a global func- The authors declare no conflict of interest. tional view of the plant kinesin superfamily. This article is a PNAS Direct Submission. The moss Physcomitrella patens is an emerging model land 1To whom correspondence should be addressed. E-mail: [email protected]. plant, for which a variety of molecular techniques can be used This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PLANT BIOLOGY (12–21). It has 27 chromosomes, and the genome sequence is 1073/pnas.1311243111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1311243111 PNAS | Published online March 3, 2014 | E1053–E1061 Downloaded by guest on September 28, 2021 In this study, we attempted to identify mitotic kinesins by determining the localization of all of the endogenous kinesins during mitosis of caulonemal cells in P. patens.Among60 kinesins for which expression was detected, 43 exhibited chro- mosome, spindle, or phragmoplast localization. Surprisingly, all but one kinesin showed a localization pattern distinct from the animal homologs. We further identified postanaphase mitotic functions of kinesin-5 by using RNAi. Results Overview of P. patens Kinesins. In this article, we followed the nomenclatures proposed by Shen et al. (28), who surveyed kinesin gene sequences in the P. patens genome and named individual kinesins based on ref. 1. Our independent BLAST search was ba- sically consistent with Shen et al. For example, kinesin-1, -3, -6, -10, and -11 were missing in the genome of P. patens. However, we additionally found two kinesin-like genes that were not listed in (28) (we named them kinesin 12-Ip and kinesin 12-IId). In total, we identified 78 kinesins, 69 of which could be classified into 15 subfamilies (kinesins 1–14 and kinesin-ARK). The remaining nine kinesins that did not belong to any subfamilies were called “orphan” kinesins (28) (Tables S1 and S2). Endogenous Kinesin Localization During Mitosis. We attempted to place the citrine [a GFP variant (29)] gene in-frame at the C-terminal end of all 78 kinesins (Fig. 1 A and B). We chose the kinesin C terminus for Citrine tagging, because N-terminal tag- ging is technically more difficult. One caveat of our strategy, however, is that tagging of Citrine to the C terminus might perturb the kinesin structure, and therefore mislocalization may be induced. Although this may be the case, this approach enables a first-pass genome-wide approach for analyzing kinesin locali- zation. Additionally, C-terminal tagging has been successful for Fig. 1. Flowchart of mitotic kinesin localizome. Experimental procedure to many kinesins in a number of different systems (3, 6, 30–32). comprehensively determine the mitotic localization of endogenous kinesins Using PCR, we confirmed proper alteration of the kinesin loci in P. patens.(A) The mCherry-tubulin–expressing cell line was used as the via homologous recombination for 69 kinesins (Fig. 1C, Left). parental cell line. A cell in metaphase is displayed (red, mCherry-tubulin; For these, we selected two independent transgenic lines for the green, autofluorescence of chloroplasts). (B) The C terminus of the endog- localization analysis in caulonemal cells (Fig. 1D). Endogenous enous kinesin gene was tagged with the citrine gene by homologous re- tagging could not been confirmed for three kinesins (kinesin combination. The citrine gene was followed by the terminator of Agrobacterium tumefaciens nopaline synthase and the G418-resistant cassette (nptII). Black 4-Id, 12-Ij, and 12-IIb), because two sets of the PCR primers we – boxes, untranslated region; white boxes, exons; black lines, introns; red designed were not functional. For those lines, 10 13 indepen- arrows, PCR primers used for strain confirmation.
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