Male meiotic spindle features that efficiently segregate paired and lagging chromosomes

Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes to produce sperm. Using live imaging and tomographic reconstructions of spermatocyte meiotic spindles in Caenorhabditis elegans, we find the lagging X chromosome, a distinctive feature of anaphase I in C. elegans males, is due to lack of chromosome pairing. The unpaired chromosome remains tethered to centrosomes by lengthening kinetochore microtubules, which are under tension, suggesting that a ‘tug of war’ reliably resolves lagging. We find spermatocytes exhibit simultaneous pole-to-chromosome shortening (anaphase A) and pole-to-pole elongation (anaphase B). Electron tomography unexpectedly revealed spermatocyte anaphase A does not stem solely from kinetochore microtubule shortening. Instead, movement of autosomes is largely driven by distance change between chromosomes, microtubules, and centrosomes upon tension release during anaphase. Overall, we define novel features that segregate both lagging and paired chromosomes for optimal sperm production.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: We did not use a statistical method to estimate appropriate sample sizes. We recorded and analyzed 31 spindles in meiosis I and 50 spindles in meiosis II within 6 C. elegans males. The total number of spindles used in the different analyses is stated both in the main text and in the corresponding figures/figure legends. For the EM analyses, we analyzed 8 complete and 3 partial spindles from 8 individual C. elegans males.

Statistical reporting
• Statistical analysis methods should be described and justified • Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10) • For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d) • Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used Data were plotted giving the mean value with standard deviation in all cases (shown either as error bars or shaded areas). In some plots the individual data points are included to illustrate the distribution of the data. Distributions of individual microtubule populations were compared using a one-way ANOVA. Levels of significance shown are: * p <= 0.05; ** p <= 0.01; *** p <= 0.001. In Figure 8B, D and F, the comparisons among the data sets were omitted in order to maintain the 'readability' of the graphs. All comparisons for these distributions are shown in Appendix- Figure 5.
Wild-type spindles were grouped according to their respective meiotic division (either meiosis I or II), and mutant samples were grouped according to their respective genotype (only meiosis I was investigated). As given in Materials and Methods, all microtubules positioned within a ribosome-free zone (i.e. with a distance of max. 150 nm from the chromosome surface) were classified as either 'end-on' or 'lateral'. Then microtubules were grouped according to the closest chromosome. X chromosome-associated microtubules were assigned separately.
eLife Sciences Publications, Ltd is a limited liability non-profit non-stock corporation incorporated in the State of Delaware, USA, with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address 1st Floor, 24 Hills Road, Cambridge CB2 1JP | August 2014 3 • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Please indicate the figures or tables for which source data files have been provided: The EM model files for microtubules and chromosome surfaces (Amira file format) have been uploaded to the TU Dresden Open Access Repository and Archive (OpARA) and are available for download as open access under: http://dx.doi.org/10.25532/OPARA-56