KTN80 confers precision to microtubule severing by specific targeting of katanin complexes in plant cells
2017; Springer Nature; Volume: 36; Issue: 23 Linguagem: Inglês
10.15252/embj.201796823
ISSN1460-2075
AutoresChaofeng Wang, Weiwei Liu, Guangda Wang, Jun Li, Li Dong, Libo Han, Qi Wang, Juan Tian, Yanjun Yu, Caixia Gao, Zhaosheng Kong,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle4 October 2017free access Source DataTransparent process KTN80 confers precision to microtubule severing by specific targeting of katanin complexes in plant cells Chaofeng Wang State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Weiwei Liu State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Guangda Wang orcid.org/0000-0001-8938-1302 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jun Li University of Chinese Academy of Sciences, Beijing, China State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Li Dong State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Libo Han State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qi Wang State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Juan Tian State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yanjun Yu State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Caixia Gao State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zhaosheng Kong Corresponding Author [email protected] orcid.org/0000-0003-3788-7883 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chaofeng Wang State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Weiwei Liu State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Guangda Wang orcid.org/0000-0001-8938-1302 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jun Li University of Chinese Academy of Sciences, Beijing, China State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Li Dong State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Libo Han State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qi Wang State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Juan Tian State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yanjun Yu State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Caixia Gao State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zhaosheng Kong Corresponding Author [email protected] orcid.org/0000-0003-3788-7883 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Author Information Chaofeng Wang1,2,‡, Weiwei Liu1,2,‡, Guangda Wang1,2,‡, Jun Li2,3, Li Dong1, Libo Han1, Qi Wang1, Juan Tian1, Yanjun Yu1, Caixia Gao3 and Zhaosheng Kong *,1 1State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 2University of Chinese Academy of Sciences, Beijing, China 3State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel/Fax: +86 10 6480 6099; E-mail: [email protected] EMBO J (2017)36:3435-3447https://doi.org/10.15252/embj.201796823 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The microtubule (MT)-severing enzyme katanin triggers dynamic reorientation of cortical MT arrays that play crucial functions during plant cell morphogenesis, such as cell elongation, cell wall biosynthesis, and hormonal signaling. MT severing specifically occurs at crossover or branching nucleation sites in living Arabidopsis cells. This differs from the random severing observed along the entire length of single MTs in vitro and strongly suggests that a precise control mechanism must exist in vivo. However, how katanin senses and cleaves at MT crossover and branching nucleation sites in vivo has remained unknown. Here, we show that the katanin p80 subunit KTN80 confers precision to MT severing by specific targeting of the katanin p60 subunit KTN1 to MT cleavage sites and that KTN1 is required for oligomerization of functional KTN80–KTN1 complexes that catalyze severing. Moreover, our findings suggest that the katanin complex in Arabidopsis is composed of a hexamer of KTN1–KTN80 heterodimers that sense MT geometry to confer precise MT severing. Our findings shed light on the precise control mechanism of MT severing in plant cells, which may be relevant for other eukaryotes. Synopsis The Arabidopsis thaliana katanin p80 subunit KTN80 plays a crucial role in defining the site of microtubule severing by precisely targeting katanin complex to microtubule crossover and branching nucleation sites. Four KTN80 isoforms act redundantly to sever microtubules at crossover and branching nucleation sites. KTN80 targets the katanin p60 subunit KTN1 to microtubules. KTN1 is dispensable for KTN80 recruitment to microtubules. KTN1 is required for hexamerisation of KTN1–KTN80 heterodimers to form an active katanin complex. Introduction Plant cells establish cortical microtubule (MT) arrays that are independent of centrosomes and closely aligned with the plasma membrane in a two-dimensional layer (Ehrhardt, 2008; Wasteneys & Ambrose, 2009; Shaw, 2013). During cell morphogenesis, plant cortical MTs play a crucial role by providing tracks for cellulose synthase complexes in cellulose biosynthesis (Paredez et al, 2006; McFarlane et al, 2014; Watanabe et al, 2015; Li et al, 2016). Additionally, plant cortical MTs are also thought to act as sensors that respond to developmental and environmental stimuli via their dynamic reorganization (Nick, 2013). The majority of nascent MTs formed during remodeling are nucleated from the wall of the pre-existing MTs in either a branching or parallel fashion (Nakamura et al, 2010). Previously, we showed that the augmin complex recruits the γ-tubulin complex to pre-existing MTs where it triggers nascent MT nucleation (Liu et al, 2014). The fate of nascent MTs is usually associated with the MT-severing enzyme katanin complex, which severs daughter MTs at branching nucleation sites, after which the detached daughter MTs translocate via treadmilling to form new configuration of MT arrays (Nakamura et al, 2010). Katanin is composed of a 60-kDa catalytic ATPase Associated with diverse cellular Activities (AAA) subunit and an 80-kDa WD-40 repeat-containing regulatory subunit (McNally & Vale, 1993; Roll-Mecak & McNally, 2010; Sharp & Ross, 2012). In vitro, the Arabidopsis p60 subunit KTN1 (also named KSS, Katanin Small Subunit) severs MTs along their entire length (Stoppin-Mellet et al, 2002, 2007). Negative staining electron microscopy showed that KTN1/KSS forms a hexamer ring structure (Stoppin-Mellet et al, 2007), consistent with the higher-order structure of its sea urchin homolog and another MT-severing enzyme, spastin (Hartman et al, 1998; Hartman & Vale, 1999; Roll-Mecak & Vale, 2008), through the conserved AAA+ domain in the presence of ATP. Notably, genetic studies have shown that KTN1 plays crucial roles in cell morphogenesis, cell wall mechanics, and hormonal signaling (Burk et al, 2001; Webb et al, 2002; Bouquin et al, 2003; Uyttewaal et al, 2012; Peaucelle et al, 2015). Intriguingly, in addition to its role in severing at branching nucleation sites (Nakamura et al, 2010), KTN1 was observed to be primarily recruited to MT crossover sites to initiate severing (Lindeboom et al, 2013; Zhang et al, 2013). MT severing plays central roles in driving the dynamic remodeling of cortical MT arrays during plant cell morphogenesis and environmental adaption, affecting processes, such as hormonal signaling, phototropic responses, and mechanical stress responses (Uyttewaal et al, 2012; Lindeboom et al, 2013; Roll-Mecak, 2013; Wightman et al, 2013; Sampathkumar et al, 2014; Peaucelle et al, 2015). Unlike random severing along single MTs in vitro, live-cell observations showed that KTN1 is primarily recruited to MT crossovers and branching nucleation sites to trigger severing (Nakamura et al, 2010; Wightman et al, 2013; Zhang et al, 2013), strongly indicating the existence of a precise control mechanism for MT severing in vivo. However, it remains to be determined how severing is precisely triggered at MT crossovers and branching nucleation sites. In particular, how the p80 subunits participate in precise MT severing and in forming the katanin supercomplex in vivo has not yet been explored. Hence, the p80 subunit is the primary candidate for uncovering the enigma of precise MT severing. Here, we use live-cell imaging, genetic and biochemical approaches, to investigate the in vivo function of katanin p80 subunits in MT severing. We find that the p80 subunit is required for specific targeting of the p60 subunit to sites of MT severing. We also find that de novo assembly of the p60-p80 multimeric katanin supercomplex on MTs confers precise MT severing at crossover sites and branching nucleation sites in living Arabidopsis cells. Results Four KTN80s act redundantly to regulate anisotropic cell elongation during plant cell morphogenesis The Arabidopsis thaliana genome has four loci encoding katanin p80 subunits, At1G11160, At1G61210, At5G08390, and At5G23430, which we designated the encoded katanin subunits KTN80.1, 2, 3, and 4, respectively. Among these, KTN80.1 and KTN80.2 fall into one subclade in the phylogenetic tree, and KTN80.3 and KTN80.4 belong to another subclade (Fig EV1A). Previous mass spectrometry assays identified KTN80.2 and KTN80.3 in microtubule-associated protein-enriched preparations (Hamada et al, 2013), and microarray analyses detected the transcripts of all four KTN80s (identified from the AtGenExpress database, http://jsp.weigelworld.org/expviz/expviz.jsp). KTN80.3 and KTN80.4 showed similar expression patterns, with high expression levels, whereas KTN80.1 and KTN80.2 showed relatively low expression levels (based on data from the AtGenExpress database). We further validated the expression patterns of the KTN80s by RT–PCR (Fig EV1B and C), and the results were consistent with the microarray data. Collectively, these results indicate that the four Arabidopsis KTN80s are expressed in all tissues examined and KTN80s may play redundant roles during development, together with the single p60 subunit encoded by KTN1. Click here to expand this figure. Figure EV1. All four KTN80s were detected in the tissues examined but showed different expression patterns Phylogenetic tree of KTN80s in Arabidopsis thaliana (At), Oryza sativa (Os), Zea mays (Zm), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), and Homo sapiens (Hs). The tree was constructed by the maximum-likelihood method using Mega 5 software with 1,000 bootstrap replicates. RT–PCR of KTN80s and KTN1 in different tissues. Fr, fruit; Fl, flower; L, leaf; S, stem; R, root. The ubiquitously expressed UBQ5 (At3G62250) gene was used as the reference. Relative intensity of gene expression in (B). The gel analyzer tool in ImageJ software was employed for intensity calculations. Download figure Download PowerPoint To gain genetic insights into the in vivo function of KTN80s during development, we generated stable ktn80.1 ktn80.2 (termed ktn80.12) and ktn80.3 ktn80.4 (termed ktn80.34) double mutants using CRISPR/Cas9 genome editing technology (Fig 1A and B) (Wang et al, 2014). Those double mutants were further confirmed to be null because the frameshift insertions/deletions in the coding sequence resulted in premature stop codons (Fig 1C). However, the ktn80.12 and ktn80.34 double mutants grow normally, and they do not show obvious growth defects (Fig 1A and B). Thus, we next created ktn80.1234 quadruple mutants by crossing the ktn80.12 and ktn80.34 double mutants (Fig 1A and B). Notably, the ktn80.1234 quadruple mutants exhibited a severe dwarf phenotype, with smaller, rounder dark-green rosette leaves and wider, shorter petioles compared to wild type, as was also seen for the katanin p60 mutant lue1 (Fig 1A and B). The lue1 mutant is confirmed to be a KTN1 null allele, which contains a nonsense mutation in the fifth exon (at the 1,179-bp position), very close to the T-DNA insertion site in the ktn1-2 null mutant (Bouquin et al, 2003; Nakamura et al, 2010). Collectively, the results indicate that the four KTN80s redundantly regulate anisotropic elongation during plant cell morphogenesis, and suggest that KTN80s function together with the p60 subunit KTN1 during development. Figure 1. Generation, genetic identification, and phenotypic analysis of the ktn80.1234 quadruple mutants A, B. Phenotypic comparison of the wild-type control (Col-0), various KTN80 knockout lines, and the lue1 mutant. The ktn80.12 and ktn80.34 double mutants and ktn80.1234 quadruple mutants were obtained via CRISPR/Cas9 genome editing technology. The order of representative rosette leaves shown in (B) is same as that in (A). C. The gene structures of KTN80s and the respective edited sites. The 5′-UTR and 3′-UTR are shown in light blue, CDS are shown in dark blue, boxes indicate exons, and lines indicate introns. Red bars in exons indicate the positions of sgRNA:Cas9 targets. The sgRNA:Cas9 targets are highlighted in red boxes, and the mutated sites are shown in red. Download figure Download PowerPoint KTN80s function in precise MT severing at crossovers and branching nucleation sites To explore whether the KTN80s contribute to MT severing, we made KTN80-GFP fusion constructs using the KTN80 genomic sequences, including the upstream promoter region, and found that these constructs fully complemented the ktn80.1234 quadruple mutant (Fig EV2A). We next performed live-cell imaging using dual-labeled transgenic lines expressing both KTN80-GFPs and mCherry-TUB6 (β-tubulin 6, for labeling cortical MTs). In leaf epidermal pavement cells, KTN80s decorated the cortical MTs and appeared as discrete particles that remained immobile for various lengths of time before disappearing from the cortical MTs (Movies EV1 and EV2). Notably, most KTN80 particles specifically localized to either MT crossovers (~52%) or branching nucleation sites (~20%), and half led to successful MT severing at these sites (Fig 2A–E, Movies EV1 and EV2). The distribution and dynamic behavior of the KTN80s were comparable to those of the control p60 subunit KTN1 (Fig 2E and F), as recorded using a functional fusion of GFP-KTN1 that fully complements the lue1 mutant phenotype (Fig EV2B). We found that severing of single MTs was negligible, with a frequency of approximately 1% for both KTN80s and KTN1 (Fig 2E). Taken together, these results indicate that KTN80s specifically function in MT severing at crossover and branching nucleation sites, further suggesting that KTN80s and KTN1 control precise MT severing through the formation of functional MT-severing complexes. Click here to expand this figure. Figure EV2. Functional complementation tests of GFP-KTN1 and KTN80-GFP fusion proteins When PKTN80.1::KTN80.1-GFP, PKTN80.2::KTN80.2-GFP, PKTN80.3::KTN80.3-GFP, and PKTN80.4::KTN80.4-GFP constructs were introduced into the ktn80.1234 quadruple mutant, growth defects of the quadruple mutant were fully rescued. Expression of the PKTN1::GFP-KTN1 construct fully restores growth defects of the lue1 mutant. Download figure Download PowerPoint Figure 2. KTN80s function in MT severing at crossovers and branching nucleation sites Time-lapse images showing that KTN80.4 is associated with MT severing at crossover sites. GFP-labeled KTN80.4 particles are pseudo-colored in red, and MTs (labeled with mCherry-TUB6) are shown in green. Yellow arrows track the growing plus end of the crossing MT. Yellow arrowheads indicate KTN80.4, which dwells at MT crossover sites. Hollow arrowheads indicate the crossing MT gets severed at the crossover site and the new plus end of the severed MT undergoes shrinkage. The dotted yellow line indicates the position and direction for the kymograph shown in (C). Scale bars, 2 μm. See also Movie EV1. Time-lapse images showing that KTN80.4 is associated with MT severing at branching nucleation sites. Yellow arrows track the growing plus end of the nascent MT during branching nucleation. Yellow arrowheads indicate KTN80.4, which dwells at the branch nucleation site. Hollow arrowheads indicate the nascent MT gets severed at the branch nucleation site and the new minus end of the severed daughter MT undergoes shrinkage. The dotted yellow line indicates the position and direction for the kymograph shown in (D). Scale bars, 2 μm. See also Movie EV2. Kymograph showing the severing event shown in (A). The green slope indicated by the yellow arrow illustrates the growth of the crossing MT, and the red patch indicated by the yellow arrowhead shows the short dwell time of KTN80.4. The asterisk indicates the loss of MT signal caused by severing of the crossing MT at the crossover site and subsequent shrinking of the new plus end of the severed MT. Scale bar, 1 μm. Kymograph showing the severing event shown in (B). The green slope indicated by the yellow arrow illustrates the growth of the daughter MT of branching nucleation, and the red patch indicated by the yellow arrowhead shows the short dwell time of KTN80.4. The asterisk indicates the loss of MT signal caused by severing of the daughter MT at the branch nucleation site and subsequent shrinking of the minus end of the severed daughter MT. Scale bar, 1 μm. Distribution of katanin (red dots) at cortical MTs (green lines) and associated MT-severing events. 528 KTN80-associated events from three cells and 331 KTN1-related events from three cells were collected for analysis, respectively. Comparison of residency times of katanin during MT severing. 371 KTN80-associated events from three cells and 261 KTN1-related events from three cells were collected for analysis, respectively. Error bars indicate SEM. Source data are available online for this figure. Source Data for Figure 2 [embj201796823-sup-0011-SDataFig2.xlsx] Download figure Download PowerPoint KTN80 quadruple mutant displays a complex cortical MT network with stable entanglements To explore the cellular mechanisms associated with cell elongation defects in the ktn80.1234 quadruple mutants, we analyzed the organization of MTs in mutant cells. Epidermal pavement cells in ktn80.1234 quadruple mutant leaves displayed more complex MT networks with bent and long MTs at higher density, frequently forming stable entanglements, compared to the wild-type control (Fig 3A). Strikingly, epidermal petiole cells of the quadruple mutants exhibited net-like MT arrays, whereas the wild-type control showed well-aligned MT arrays (Fig 3B). The abnormal MT organization in the ktn80.1234 quadruple mutant is reminiscent of that observed in the p60 mutant alleles, fra2, lue1, and bot1 (Burk et al, 2001; Bouquin et al, 2003; Wightman et al, 2013). Taken together, these results indicate that the four KTN80s act redundantly to regulate cell elongation by modulating MT organization, possibly by manipulating MT severing. Figure 3. The ktn80.1234 quadruple mutants display abnormal MT organization, abolished MT severing, and disrupted recruitment of KTN1 Comparison of cortical MT (labeled with GFP-TUB6) patterns in epidermal pavement cells between the control and the ktn80.1234 quadruple mutant. Cell outlines are highlighted with green dotted lines. Scale bar, 5 μm. Comparison of cortical MT (GFP-TUB6) patterns in epidermal petiole cells (shown in part) between the control and the ktn80.1234 quadruple mutant. Scale bar, 5 μm. MT severing is completely abolished in epidermal pavement cells of ktn80.1234 quadruple mutant cells, compared with the control cells. Yellow arrows track the growing ends of the crossing MT, which forms many crossovers with other MTs. In control cells, the crossing MT gets severed at the crossover site (marked with yellow hollow arrowheads) and consequently becomes fragmented or depolymerized. Dotted yellow lines depict the intact crossing MT or MT fragments caused by severing. In ktn80.1234 quadruple mutant cells, no severing was observed, and the MT crossovers (indicated by yellow open arrowheads) keep stable for a long time. Scale bars, 5 μm. See also Movie EV3. The recruitment of GFP-labeled KTN1 (pseudo-colored in red) to MTs (mCherry-TUB6; pseudo-colored in green) is completely disrupted in ktn80.1234 quadruple mutant cells, compared with the wild-type control. In control cells, cyan arrows indicate crossover-localized KTN1, orange arrows indicate KTN1 landing at branching nucleation sites, and KTN1 on single MTs is marked with the yellow arrow. Scale bar, 2 μm. See also Movie EV4. Western blot indicates that GFP-KTN1 fusion proteins could be detected by the anti-GFP monoclonal antibodies in ktn80.1234 quadruple mutants. Coomassie Brilliant Blue (CBB) staining of SDS–PAGE gels as the loading control. Source data are available online for this figure. Source Data for Figure 3 [embj201796823-sup-0012-SDataFig3.tif] Download figure Download PowerPoint Loss of KTN80 function results in the disruption of KTN1 recruitment and consequent abolishment of MT severing To ascertain how the stable MT entanglements form, we compared MT dynamics in ktn80.1234 quadruple mutant cells and wild-type control cells. MT severing frequently occurs at either branching nucleation sites or crossover sites in control cells (Fig 3C, Movie EV3). MT severing at crossovers effectively eliminates discordant MTs in establishing a well-aligned MT array (Wightman & Turner, 2007; Wightman et al, 2013). When a single MT passes through an aligned array, many crossovers are formed. In most cases, the invading (crossing) MT gets severed at the sites where it crosses other MTs and finally becomes fragmented or depolymerized (Fig 3C, Movie EV3). By contrast, no severing was observed at MT crossover sites in ktn80.1234 quadruple mutant cells (2,612 μm2 areas in three cells were examined over 5 min) (Fig 3C, Movie EV3). Consequently, MT crossovers remain stable for a long time and they accumulate to form the MT entanglements that characterize mutant cells (Fig 3A–C). We next monitored the behaviors of KTN1 in ktn80.1234 quadruple mutant cells to gain further insight into the specific roles of KTN80 in MT severing in vivo. Notably, no GFP-KTN1 particles were observed to localize to MTs (Fig 3D, Movie EV4). We used a construct that co-expressed both GFP-KTN1 and mCherry-TUB6 (see 4), and while we observed mCherry-labeled MTs, GFP-KTN1 was not seen in ktn80.1234 quadruple mutant cells, although GFP-KTN1 expression was confirmed by Western blotting (Fig 3E). These results demonstrate that MT severing is completely abolished in the ktn80.1234 quadruple mutants due to the impaired recruitment of KTN1 to MTs (3,388 μm2 areas in four cells were examined over 5 min). These findings show that KTN80s are required to target katanin complexes to sites where MT severing occurs, and also suggest that KTN80s recruits KTN1 onto MTs during assembly of katanin supercomplexes. KTN1 is dispensable for KTN80 localization, but KTN80 is required for proper targeting of katanin complexes in vivo We next evaluated KTN80s in the lue1 mutant background to clarify the spatial and temporal relationship of KTN1 and KTN80s to MT severing. We found that KTN80s were able to localize on MTs, primarily at MT crossovers and branching nucleation sites, but never initiated MT severing in lue1 cells (Fig 4A, Movie EV5), unlike what we observed in the wild-type control (Fig 4A, Movie EV5). Quantitatively, the distribution and residency times of KTN80s puncta were comparable in lue1 and wild-type cells (Fig 4B and C). lue1 cells have more MT crossovers than do wild-type cells (Fig 4A, Movie EV5) but, after normalization, we found comparable frequencies (KTN80 puncta at MT crossovers per 100 μm2/s per crossover) of KTN80 recruitment between lue1 and control cells (Fig 4D). Although the recruitment of KTN1 to MTs depends on KTN80s (see Fig 3D), it was formally possible that KTN1 encodes a N-terminal truncated protein that interferes with or modifies katanin function in lue1 cells. Therefore, we used CRISPR/Cas9 to generate a new KTN1 null allele ktn1-c1, and we found that this new mutant shows identical growth defects, as those shown by lue1, fra2 and bot1 mutants (Fig EV3A and B). The ktn1-c1 mutant contains a frameshift mutation at 22 bp from the start codon and it only encodes 7 amino acids of the N-terminal region of KTN1 plus 14 novel amino acids (Fig EV3B), indicating that it is an absolutely KTN1 null allele. Subsequent observations further confirmed that MT severing is completely disrupted (3,134.08 μm2 area in four cells were examined over 3 min), but with normal KTN80 behaviors in ktn1-c1 cells (Fig EV3C, Movie EV6). These results show that loss of KTN1 does not affect KTN80 localization and behaviors, but KTN80 is required for KTN1 targeting, strongly indicating that KTN80 confers precision to MT severing. Figure 4. KTN1 is dispensable for KTN80 targeting and dynamics Comparison of the localization of GFP-labeled KTN80.3 (red particles) at cortical MTs (mCherry-TUB6, pseudo-colored in green) between the lue1 cells and the wild-type control. Cyan arrows indicate crossover-localized KTN80.3. Orange arrows indicate KTN80.3 landing at branching nucleation sites. In control cells, KTN80.3 triggers MT severing at crossovers (marked with cyan hollow arrowheads) and branching nucleation sites (marked with orange hollow arrowheads), respectively. In lue1 cells, MTs never get severed, and the MT configuration of the indicated crossover (cyan open arrowheads) and the branching nucleation (orange open arrowheads) keeps stable for a long time. Scale bar, 2 μm. See also Movie EV5. Comparison of the proportions of KTN80.3 that show different residences at three representative sites, between lue1 cells and the control. 514 KTN80.3-associated events in three wild-type cells and 372 KTN80.3-associated events in three lue1 cells were collected for analysis both (B) and (C). Error bars indicate SD. Comparison of residency times of KTN80.3 particles, which are distributed at three representative sites, cortical MT between lue1 and control cells. Error bars indicate SEM. Comparison of relative landing frequency (expressed as observed landings per 100 μm2/s per crossover) of KTN80.3, which localizes at MT crossover sites, between lue1 and control cells; 350 KTN80.3-associated events were detected in 3,225 μm2 areas of three wild-type cells over 12 min, and 225 KTN80.3-associated events were detected in 1,637 μm2 areas of three lue1 cells over 10 min. Error bars indicate SD. Source data are available online for this figure. Source Data for Figure 4 [embj201796823-sup-0013-SDataFig4.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. KTN80 behaviors in a new KTN1 null allele, ktn1-c1 Comparison of the growth phenotype between the wild-type control and ktn1-c1, which was obtained via CRISPR/Cas9 genome editing technology. The schematic diagram illustrates the gene structures of KTN1 and the mutation sites of the ktn1-c1, lue1, and ktn1-2 mutant alleles, respectively. Boxes indicate exons (gray, untranslated regions; black, the coding region), and thick lines indicate introns. The sgRNA:Cas9 targets are highlighted in the
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