Kinetochore dynein is sufficient to biorient chromosomes and remodel the outer kinetochore

Multiple microtubule-directed activities concentrate on chromosomes during mitosis to ensure their accurate distribution to daughter cells. These activities include couplers and dynamics regulators localized at the kinetochore, the specialized microtubule interface built on centromeric chromatin, as well as motor proteins recruited to kinetochores and to mitotic chromatin. Here, we describe an in vivo reconstruction approach in which the effect of removing the major microtubule-directed activities on mitotic chromosomes is compared to the selective presence of individual activities. This approach revealed that the kinetochore dynein module, comprised of the minus end-directed motor cytoplasmic dynein and its kinetochore-specific adapters, is sufficient to biorient chromosomes and to remodel outer kinetochore composition following microtubule attachment; by contrast, the kinetochore dynein module is unable to support chromosome congression. The chromosome-autonomous action of kinetochore dynein, in the absence of the other major microtubule-directed factors on chromosomes, rotates and orients a substantial proportion of chromosomes such that their sister chromatids attach to opposite spindle poles. In tight coupling with orientation, the kinetochore dynein module drives removal of outermost kinetochore components, including the dynein motor itself and spindle checkpoint activators. The removal is independent of the other major microtubule-directed activities and kinetochore-localized protein phosphatase 1, suggesting that it is intrinsic to the kinetochore dynein module. These observations indicate that the kinetochore dynein module has the ability coordinate chromosome biorientation with attachment state-sensitive remodeling of the outer kinetochore that facilitates cell cycle progression.

. The Control and Blank Canvas curves are the same as in Fig. 1D and are plotted to aid comparison. n is number of embryos analyzed. (D) Examples of anaphase segregation defects observed in the absence of the kinetochore dynein module. Scale bar, 5 µm. (E) Summary of missegregation events observed in anaphase of one-cell embryos. n is number of embryos imaged. Inhibition of the spindle checkpoint does not explain the segregation defect observed in the absence of the kinetochore dynein module.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ;https://doi.org/10.1101/2023 doi: bioRxiv preprint Figure S3. Imaging of GFP::SPDL-1, ROD-1::mScarlet and GFP::MAD-1, and supporting data for analysis of Ndc80 module and protein phosphatase 1 inhibitions.

(A) & (B)
Images of in situ-tagged GFP::SPDL-1 and  embryos. As the mutant NDC-80 transgene was not present, this condition is labeled Dynein Only*.
SPDL-1 behaved similarly to DHC-1, in that kinetochore-autonomous removal was observed. However, ROD-1 behaved distinctly-its levels were maintained at oriented kinetochores. Scale bars, 1 µm in (A) and 5 µm in (B). (C) Image sequence of GFP::MAD-1 in the kinetochore dynein only state. The high signal of GFP::MAD-1 in the spindle region, together with its later recruitment, made imaging its dynamics on single kinetochores challenging. Nonetheless, chromosomes with clear kinetochore MAD-1 signal exhibited orientation-coupled removal from kinetochores. Scale bar, 1 µm. (D) Interval between the Before and After timepoints analyzed in Fig. 5A. The same interval is also plotted for the condition analyzed in Fig. 5B, although DHC-1 signal intensity before orientation was not measured. (E) Plot of DHC-1::GFP signal on persistently lateral chromosomes after anaphase onset for the indicated conditions. Error bars are the 95% confidence interval.
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METHODS:
C. elegans strains C. elegans strains are described in Table S1. All strains were maintained using standard C. elegans growth media and imaged at 20ºC. Endogenous locus GFP tagging was performed using CRISPR/Cas9 53 at the dhc-1 locus (dynein heavy chain) and the klp-19 locus (chromokinesin). For both dhc-1::gfp and gfp::klp-19 the repair template contained two homology arms, a linker sequence (GGRAGSG) and a sequence encoding GFP. GFP integrations were confirmed by PCR. For details on gRNAs, see Table S2.

RNA-mediated interference
DNA templates were generated via PCR using the primers as specified in Table S3, and subsequently purified using a QIAquick PCR Purification Kit (Qiagen). Single-stranded RNA was generated from each DNA template using a MEGAscript TM T3 and T7 Transcription Kit (Invitrogen), and subsequently purified using a MEGAclear TM Transcription Clean-Up Kit (Invitrogen). Double-stranded RNA (dsRNA) was generated by annealing the single-stranded RNAs at 37°C for 30 minutes 43 . 36-46h before dissection and embryo imaging, the dsRNA was injected into L4 hermaphrodites, which were maintained at 20°C. All RNAi experiments were performed using 1 mg/ml individual dsRNAs, or 1:1 or 1:1:1 mixtures of 1 mg/ml individual dsRNAs.

One-cell embryo fluorescence microscopy and image analysis
One-cell embryos were dissected from adult hermaphrodites in M9 buffer, placed onto a microscope slide containing a 2% agarose pad, and subsequently covered with a 22x22 mm highprecision cover glass (No. 1.5H, Marienfeld).
Embryos were imaged on a spinning-disk confocal (Revolution XD Confocal System; Andor Technology) with a confocal scanner unit (CSU-10, Yokogawa Corporation) attached to an inverted microscope body (TE2000-E, Nikon), illuminated using solid-state 100 mW lasers using either a 60X or 100X 1.4 NA Plan Apochromat oil objective (Nikon), and an EMCCD camera (iXon DV887, Andor Technology) (Desai Lab, San Diego).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ; https://doi.org/10. 1101/2023 For localization analysis of NDC-80::GFP, GFP::KLP-19 and DHC-1::GFP, 5 x 1.5 µm z-stacks were acquired every 10s and for KNL-1::GFP every 3s. For single chromosome localization analysis of DHC-1::GFP, 5 x 1.5 µm z-stacks were acquired every 3 s starting ~1 min after NEBD and maximum intensity projections (MIPs) generated using Image J (Fiji). Subsequently, the fluorescent background was subtracted and a rectangular box (0.4 x 1.6 µm) fitted adjacent to the mCherry::H2B signal (chromosome) encapsulating kinetochore dynein to obtain the average DHC-1::GFP intensity.
For minimum bounding box (MMB) analysis, 5 x 1.5 µm z-stacks were acquired every 3 s, MIPs generated using Image J (Fiji) and rotated to position the spindle poles horizontally. Fluorescence intensity for all MIPs in the series was normalized, converted to 8-bit and the fluorescence background subtracted. Subsequently, to each MIP image a MMB was fitted (pixel value > 0, i.e. fluorescent signal from GFP::H2B) to measure chromosome positioning.
For chromosome orientation analysis, 5 x 1.5 µm z-stacks were acquired every 2 s for GFP::H2B or 3 s for DHC-1::GFP, MIPs generated using Image J (Fiji) and rotated to position the spindle poles horizontally. Subsequently, chromosome angles were determined by fitting a line along each chromosome axis and measuring the smallest angle between the chromosome axis and spindle poleto-pole axis.
For EBP-2 imaging, a single z-section was acquired every 800 ms using 100 ms exposure, starting 1 min after NEBD. Kymographs were generated from a 5-pixel width line drawn pole-to-pole using KymographClear 54 . Summed-intensity projection (SIP) images were generated by adding 38 EBP-2 frames starting 1 min after NEBD and subtracting the background signal. Intensity profiles were generated from these SIPs, by drawing a rectangular box (1.5 x 22 µm) encapsulating both spindle poles and averaging of the pixel intensities of each column within.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   96,100,125,144,155AAAAAA)  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  ltSi711[pDC267;96,100,125,144,155AAAAAA reencoded; 96,100,125,144,155AAAAAA)    . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023