in this study ,  wild - type  refers to n2 ( bristol ) worms , while the term  control  refers to control(rnai ) worms . for most experiments , integrated lines expressing gfp::tubulin ; gfp::histone ( strain eu1067 , gift of bruce bowerman ) or gfp::tubulin ; mcherry::histone ( strain od57 , gift of jon audhya)35 were used .
emb-27(g48 ) worms obtained from the c. elegans genetics center were crossed with eu1067 to create strain av335 ( emb-27(g48)ii ; unc-119(ed3 ) ruis32[unc-119(+ ) pie-1::gfp::h2b]iii ; ruis57[unc-119(+ ) pie-1::gfp::tubulin ] ) ; these worms were used for all experiments requiring an apc - arrest .
metaphase i arrest was achieved by shifting adult av335 worms from 20c to 25c for 57 hours before imaging .
individual rnai clones picked from an rnai feeding library36 , 37 were used to inoculate overnight lb/100 g / ml amp cultures .
these saturated cultures were used to seed ngm plates containing 100 g / ml amp and 1 mm iptg ; plates were left at room temperature overnight to induce dsrna formation .
the next day , l1 worms synchronized by bleaching were put on the plates , and the worms were grown to adulthood by placing the plates at 15c for 5 days .
immunofluorescence was performed as previously described8 , 38 , using a fixation time of 30 minutes in 20c methanol .
primary antibodies used were mouse anti--tubulin ( dm1 , sigma ) at 1:500 , rabbit anti - klp-1910 at 1:4000 , rabbit anti - air-211 at 1:250 , rabbit anti - knl-339 at 1:4000 , rat anti - him-107 at 1:500 and rabbit anti - aspm-1 at 1:4000 .
anti - aspm-1 antibody ( a gift from arshad desai ) was raised and affinity purified against amino acids 9165 .
secondary antibodies used were alexa fluor 488-conjugated goat anti - mouse and alexa fluor 555-conjugated goat anti - rabbit , both at 1:400 . for live imaging , gfp- or mcherry - expressing worms
worms were picked into a drop of m9 containing 0.1% tricaine , 0.01% tetramisole40 , a coverslip was laid on top and the slide was imaged immediately .
deltavision deconvolution microscopy with a 100 objective was used for both live and fixed imaging .
image stacks were obtained at 0.2 m z steps for fixed samples and 0.3 m for live imaging , deconvolved and projected for presentation . except where noted ,
images are full projections of data stacks encompassing the entire meiotic spindle . in some cases partial projections are displayed to highlight particular features of spindle organization .
control and rnai worms were processed in parallel and image exposure times were kept constant within each experiment .
to quantitatively compare the fluorescence intensities of microtubules ensheathing the chromosomes with fluorescence adjacent to the bivalent ends , individual pixel intensities within a 4 pixel by 3334 pixel area spanning the ensheathing microtubule bundles in the vicinity of the bivalent ends were determined and graphed using softworx ( applied precision ) software ( fig .
pixel intensities in the domains outside the ensheathing microtubule bundles were averaged for each image , and these average values were then subtracted from the dataset .
these subtracted values were used to calculate ratios between the peak intensity of each ensheathing bundle and the region of minimum intensity between the bundles .
for quantification of bivalent orientation , worms expressing gfp::histone and gfp::tubulin were ethanol - fixed by picking worms onto a slide and adding 100% ethanol and letting it evaporate ( this ethanol step was performed three times before adding mounting media ) .
for monopolar spindles ( generated by klp-18 rnai ) , chromosome orientation was determined by tracing a line from the center of each aster through the midpoint of each bivalent . a second line was then drawn mirroring the long axis of each bivalent , and the angle between the two lines was measured ( fig .
, bivalents were classified either as oriented or not oriented ; bivalents were considered to be oriented if their long axes were parallel with the spindle axis . for quantification of rnai phenotypes , apc - arrested worms expressing gfp::histone and gfp::tubulin were mounted live and visualized using a 63 objective on a zeiss axioplan ii microscope .
although the apc arrest conditions yield a row of embryos arrested at metaphase i , only the most recently fertilized embryo from each gonad arm ( the + 1 embryo , next to the spermatheca ) was analyzed to avoid the possibility of problems introduced by prolonged arrest .
for quantitation of chromosome congression , only embryos that had assembled bipolar spindles were considered .
a subset of + 1 embryos in both control and experimental worms had disorganized microtubule arrays , presumably reflecting earlier stages of spindle assembly . for klp-19(rnai ) , the fractions of embryos in this excluded class were similar to control worms in both experiments ( experiment 1 : control 51.1% , klp-19(rnai ) 48% , p=0.77 .
icp-1(rnai ) embryos had an increase in disorganized arrays , possibly reflecting additional roles for icp-1 ( experiment 1 : control 51.1% , icp-1(rnai ) 61.7% , p=0.15 .
experiment 2 : control 29% , icp-1(rnai ) 52.9 % , p=0.0004 ) . in experiments assessing chromosome positions on monopolar spindles , all
+ 1 embryos in both control ( klp-18(rnai ) ) and experimental ( klp-18(rnai ) ; klp-19(rnai ) ) worms exhibited clear aster - like monopolar spindles , suggesting that monopolar spindles form immediately following initiation of the meiotic divisions .
in this study ,  wild - type  refers to n2 ( bristol ) worms , while the term  control  refers to control(rnai ) worms . for most experiments , integrated lines expressing gfp::tubulin ; gfp::histone ( strain eu1067 , gift of bruce bowerman ) or gfp::tubulin ; mcherry::histone ( strain od57 , gift of jon audhya)35 were used .
emb-27(g48 ) worms obtained from the c. elegans genetics center were crossed with eu1067 to create strain av335 ( emb-27(g48)ii ; unc-119(ed3 ) ruis32[unc-119(+ ) pie-1::gfp::h2b]iii ; ruis57[unc-119(+ ) pie-1::gfp::tubulin ] ) ; these worms were used for all experiments requiring an apc - arrest .
metaphase i arrest was achieved by shifting adult av335 worms from 20c to 25c for 57 hours before imaging .
individual rnai clones picked from an rnai feeding library36 , 37 were used to inoculate overnight lb/100 g / ml amp cultures .
these saturated cultures were used to seed ngm plates containing 100 g / ml amp and 1 mm iptg ; plates were left at room temperature overnight to induce dsrna formation .
the next day , l1 worms synchronized by bleaching were put on the plates , and the worms were grown to adulthood by placing the plates at 15c for 5 days .
immunofluorescence was performed as previously described8 , 38 , using a fixation time of 30 minutes in 20c methanol .
primary antibodies used were mouse anti--tubulin ( dm1 , sigma ) at 1:500 , rabbit anti - klp-1910 at 1:4000 , rabbit anti - air-211 at 1:250 , rabbit anti - knl-339 at 1:4000 , rat anti - him-107 at 1:500 and rabbit anti - aspm-1 at 1:4000 .
anti - aspm-1 antibody ( a gift from arshad desai ) was raised and affinity purified against amino acids 9165 .
secondary antibodies used were alexa fluor 488-conjugated goat anti - mouse and alexa fluor 555-conjugated goat anti - rabbit , both at 1:400 .
for live imaging , gfp- or mcherry - expressing worms were mounted on mp multitest slides .
worms were picked into a drop of m9 containing 0.1% tricaine , 0.01% tetramisole40 , a coverslip was laid on top and the slide was imaged immediately .
deltavision deconvolution microscopy with a 100 objective was used for both live and fixed imaging .
image stacks were obtained at 0.2 m z steps for fixed samples and 0.3 m for live imaging , deconvolved and projected for presentation . except where noted ,
images are full projections of data stacks encompassing the entire meiotic spindle . in some cases partial projections are displayed to highlight particular features of spindle organization .
control and rnai worms were processed in parallel and image exposure times were kept constant within each experiment . to quantitatively compare the fluorescence intensities of microtubules ensheathing the chromosomes with fluorescence adjacent to the bivalent ends , individual pixel intensities within a 4 pixel by 3334 pixel area spanning the ensheathing microtubule bundles in the vicinity of the bivalent ends were determined and graphed using softworx ( applied precision ) software ( fig .
pixel intensities in the domains outside the ensheathing microtubule bundles were averaged for each image , and these average values were then subtracted from the dataset .
these subtracted values were used to calculate ratios between the peak intensity of each ensheathing bundle and the region of minimum intensity between the bundles .
for quantification of bivalent orientation , worms expressing gfp::histone and gfp::tubulin were ethanol - fixed by picking worms onto a slide and adding 100% ethanol and letting it evaporate ( this ethanol step was performed three times before adding mounting media ) .
for monopolar spindles ( generated by klp-18 rnai ) , chromosome orientation was determined by tracing a line from the center of each aster through the midpoint of each bivalent .
a second line was then drawn mirroring the long axis of each bivalent , and the angle between the two lines was measured ( fig .
bivalents were classified either as oriented or not oriented ; bivalents were considered to be oriented if their long axes were parallel with the spindle axis . for quantification of rnai phenotypes , apc - arrested worms expressing gfp::histone and gfp::tubulin were mounted live and visualized using a 63 objective on a zeiss axioplan ii microscope .
although the apc arrest conditions yield a row of embryos arrested at metaphase i , only the most recently fertilized embryo from each gonad arm ( the + 1 embryo , next to the spermatheca ) was analyzed to avoid the possibility of problems introduced by prolonged arrest .
for quantitation of chromosome congression , only embryos that had assembled bipolar spindles were considered .
a subset of + 1 embryos in both control and experimental worms had disorganized microtubule arrays , presumably reflecting earlier stages of spindle assembly . for klp-19(rnai ) , the fractions of embryos in this excluded class were similar to control worms in both experiments ( experiment 1 : control 51.1% , klp-19(rnai ) 48% , p=0.77 .
icp-1(rnai ) embryos had an increase in disorganized arrays , possibly reflecting additional roles for icp-1 ( experiment 1 : control 51.1% , icp-1(rnai ) 61.7% , p=0.15 .
experiment 2 : control 29% , icp-1(rnai ) 52.9 % , p=0.0004 ) . in experiments assessing chromosome positions on monopolar spindles , all
+ 1 embryos in both control ( klp-18(rnai ) ) and experimental ( klp-18(rnai ) ; klp-19(rnai ) ) worms exhibited clear aster - like monopolar spindles , suggesting that monopolar spindles form immediately following initiation of the meiotic divisions .
 although centrosomes serve to organize microtubules in most cell types , oocyte spindles form and mediate meiotic chromosome segregation in their absence . here , we use high - resolution imaging of both bipolar and experimentally - generated monopolar spindles in c. elegans to reveal a surprising organization of microtubules and chromosomes within acentrosomal structures . 
 we find that homologous chromosome pairs ( bivalents ) are surrounded by microtubule bundles running along their sides , whereas microtubule density is extremely low at chromosome ends despite a concentration of kinetochore proteins on those regions . 
 further , we find that the chromokinesin klp-19 is targeted to a ring around the center of each bivalent and provides a polar ejection force required for congression . together , 
 these observations create a new picture of chromosome / microtubule association in acentrosomal spindles and reveal a mechanism by which metaphase alignment can be achieved utilizing this organization . 
 specifically , we propose that : 1 ) ensheathment by lateral microtubule bundles places spatial constraints on the chromosomes , thereby promoting biorientation , and 2 ) localized motors mediate movement along these bundles , thereby promoting alignment . 
Methods
Strains
RNAi
Immunofluorescence
High-resolution imaging
Quantification
Supplementary Material