- + α β αβ-tubulin heterodimer protofilament X 13 microtubule seam

- + α β αβ-tubulin heterodimer protofilament X 13 microtubule seam
Fig.1. Microtubule structure. Microtubules are composed of tubulin αβ-heterodimers. Tubulin dimers polymerize in a head-to-tail mode and produce a protofilament. In microtubule, protofilaments interact with each other by α-α and β-β tubulins, except in one pair of protofilaments, forming the seam. β-tubulin is exposed at the dynamic plus end of microtubule and α-tubulin is at the less dynamic minus end of microtubule.

+ - + - kinetochore microtubule kinetochore + + + + + + + + + - + - +
astral microtubule centrosome + + polar microtubule chromosome + - plus end motor direction of microtubule sliding + - Fig.2. Spindle organization. The spindle is composed of three populations of microtubules; astral, polar and kinetochore microtubules. Minus ends of all microtubules are at the centrosomes. Plus ends of astral microtubules are directed towards the cell cortex. Plus ends of kinetochore microtubules are at kinetochores. Plus ends of polar microtubules interact with chromosome arms and/or overlap with plus ends of polar microtubules emanating from the opposite pole in an anti-parallel fashion. Microtubules within the spindle interact with each other via microtubule associated proteins (MAPs). Here shown an example of a kinesin-5 bundling and sliding anti-parallel polar microtubules.

MITOTIC CELL centrosome microtubule direction of centrosome migration Nuclear envelope breakdown Microtubule assemble predominantly from centrosomes Separation of centrosomes condensing chromosome Bipolarity established Microtubule stabilization kinetochore Spindle assembled Microtubule stabilization - - Fig.3. Centrosomes determine bipolarity of the spindle. Microtubules are nucleated by two centrosomes and form two asters. The asters separate and determine the bipolarity of the future spindle. As the nuclear envelope disassembles, microtubules penetrate the cytoplasm, including the area of chromosomes. Capture of kinetochores by microtubules reduces the number of astral microtubules. Gradually, more microtubules interact with chromosomes, resulting in a structure of a bipolar spindle.

INTERPHASE MITOSIS importin-cargo RanGDP importin-cargo importin-SAF cargo RanGTP importin SAF NEB RanGTP RCC1 importin RanGTP RanGTP RCC1 RanGDP RanGDP RanGDP RanGDP RanGDP Fig.4. A model of Ran-GTP function in chromosome-driven spindle assembly. RCC1, bound to chromosomes, exchanges RanGDP (red) into RanGTP (yellow). In the nucleus, RanGTP outcompetes cargoes from importins. After nuclear envelope breakdown (NEB), RanGTP gradient forms. High concentration of RanGTP in the area of chromosomes releases spindle assembly factors (SAFs) from importins. Local concentration of SAFs promotes spindle assembly in the vicinity of chromosomes.

CPC: Aurora B, Incenp, Survivin/Deterin, Dasra/Borealin
inner centromere CPC: Aurora B, Incenp, Survivin/Deterin, Dasra/Borealin centromere Cenp-A inner kinetochore Cenp-C / CCAN: Cenp-C, Cenp-H, Cenp-I, Cenp-K-U Mis12 complex: Mis12, Nsl1R, Nnf1R, Dsn1/KNL-3 outer kinetochore and fibrous corona Spc105/KNL1 Ndc80 complex: Ndc80/Hec1, Nuf2, Spc24 and Spc25 SAC, dynein, Cenp-E, Mal3/EB1, Nup , others microtubules Fig.5. Kinetochore structure and composition. Kinetochore joints centromeric chromatin with microtubules. Kinetochore is assembled onto centromeric chromatin in a layered fashion. The layers, inner and outer kinetochore, as well as fibrous corona are composed of different protein complexes, which undergo hierarchical assembly. Roughly, kinetochore complexes closest to centromere are upstream of proteins located further towards microtubules. The figure also shows a region between sister kinetochore, called the inner centromere and depicts its key components.

a e b d c - + + - growth shrinkage chromosome movement Cenp-E/Cenp-meta Kinetochore-localized microtubule destabilizers eg. MCAK Tubulin dimers Fig.6. A model for chromosome positioning within the mitotic spindle. (a) kinetochore establishes lateral attachments with a microtubule and chromosome is transported towards a spindle pole. (b) close to the pole, kinetochore establishes end-on attachments with microtubules. (c) sister kinetochore is attached by a microtubule from the opposite pole and is pulled towards spindle equator. Movement of chromosome towards equator is associated with plus end microtubule depolymerization at the leading kinetochore and polymerization at the lagging kinetochore, while Cenp-E/Cenp-meta maintains association of kinetochore with microtubules. (d) congressed chromosome. (e) Cenp-E/Cenp-meta transports kinetochore of a mono-oriented chromosome along microtubule of an already positioned chromosome.

MOUSE OOCYTE Drosophila OOCYTE After NEB chromosomes microtubule aster (MTOC) Microtubule assemble around chromosomes microtubule network central spindle re-organizaion elongation Bipolarity established Fig.7. Assembly of the acentrosomal spindle. In oocytes, spindle microtubules assemble in the area of chromosomes. In mouse, microtubules are nucleated by prominent microtubule organizing centres (MTOCs). Microtubules and MTOCs interconnect in the area of chromosomes and re-arrange to form a barrel-shaped bipolar spindle, with MTOCs grouped at two poles. In Drosophila, microtubules assemble in the area of chromosomes without prominent MTOCs. Microtubules are first arranged into parallel bundles and then elongate to form a well tapered bipolar spindle.

Confirmation of disruption the functionality of kinetochore components in mutants
kmn1GD237 + + + + Dp (1;3) DC241 ; ; ♀ ♀ x ; ; ♂ ♂ FM7C + + Y + Dp (1;3) DC241 kmn1GD237 + Dp (1:3) DC241 ; ; ♂ ♂ Y + + viable = Kmn1 (Nsl1R) not expressed w + cal1MB04866 w + Df (3R) Exel6176 ; ; ♀ ♀ x ; ; ♂ ♂ w + TM6C, Sb Y + TM6B w + cal1MB04866 ; ; ♀ ♀ w + Df Exel6176 lethal = Cal1 not expressed w + mis12f03756 w + Df (3L) BSC374 ; ; ♀ ♀ x ; ; ♂ ♂ w + TM6B Y + TM6C w + mis12f03756 ; ; ♀ ♀ w + Df BSC374 lethal = Mis12 not expressed + c-met∆ + w Df (2L) Ex6028 + ; ; ♀ ♀ x ; ; ♂ ♂ + Cyo + Y Cyo + + c-met∆ + ; ; ♀ ♀ w Df (2L) Ex6028 + lethal = Cenp-meta not expressed

Recombination of fluorescent markers and nanos GAL4 driver on the third chromosome
w + nanos GAL4 [w+m] RCC1::mCherry [w+m] w + Mis12::GFP [w +m] ; ; x ♂ ♂ ; ; Y + TM6C w + TM6C ♀ ♀ w + nanos GAL4 [w+m] RCC1::mCherry [w+m] w + Pr ; ; ♀ ♀ x ; ; ♂ ♂ w + Mis12::GFP [w +m] Y + TM6B w + nanos GAL4 [w+m] RCC1::mCherry [w +m] Mis12::GFP [w+m] w + Pr ; ; x ; ; Y + ♂ ♀ ♀ TM6B w + TM6B w + nanos GaL4 [w+m] RCC1::mCherry [w +m] Mis12::GFP [w+m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6B Recombination of fluorescent markers on the second chromosome w Mis12::GFP [w +m] + w RCC1::mCherry [w +m] + ; ; ♀ ♀ x ; ; ♂ ♂ w Cyo + Y Cyo + w Mis12::GFP [w +m] + w Sco + ; ; ♀ ♀ x ; ; ♂ ♂ w RCC1::mCherry [w +m] + Y Cyo + w Mis12::GFP [w +m] RCC1::mCherry [w+m] + ; ; ♀ ♀ ♂ ♂ Y/w Cyo + Confirmation of recombination w Mis12::GFP RCC1::mCherry + w + nanos GAL4 ; ; ♂ ♂ x ; ; ♀ ♀ Y Cyo + w + TM6B w Mis12::GFP RCC1::mCherry nanos GAL4 ; ; ♀ ♀ w + + expression of markers confirmed by microscopy

Recombination of a single fluorescent marker and GAL4 mat α-tub (V37) driver on the third chromosome
w + GAL4 mat α-tub [w+m] w + GFP-CID [w +m] ; ; ♀ ♀ x ; ; ♂ ♂ w + GAL4 mat α-tub [w+m] Y + GFP-CID [w +m] w + GAL4 mat α-tub [w+m] w + Pr ; ; ♀ ♀ x ; ; ♂ ♂ w + GFP-CID [w +m] Y + TM6B w + GAL4 mat α-tub [w+m] GFP-CID [w+m] w + Df(3R)BSC318 ; ; ♂ x ; ; ♀ ♀ Y + TM6B w + TM6C w + GAL4 mat α-tub [w+m] GFP-CID [w+m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6C w + GAL4 mat α-tub [w+m] w + GFP-Rod [w +m] ; ; ♀ ♀ x w + GAL4 mat α-tub [w+m] ; ; ♂ ♂ Y + GFP-Rod [w +m] w + GAL4 mat α-tub [w+m] ; ; ♀ ♀ x w + Pr w + GFP-Rod [w +m] ; ; ♂ Y + TM6B w + GAL4 mat α-tub [w+m] GFP-Rod [w+m] w + Df(3R)BSC318 ; ; ♂ x ; ; ♀ ♀ Y + TM6B w + TM6C w + GAL4 mat α-tub [w+m] GFP-Rod [w+m] Y/w ; ; + TM6C ♀ ♀ ♂ ♂ w + GAL4 mat α-tub [w+m] w + Nsl1R::GFP [w +m] ; ; ♀ ♀ x ; ; w + GAL4 mat α-tub [w+m] Y + TM6C ♂ ♂ w + GAL4 mat α-tub [w+m] w + Pr ; ; ♀ ♀ x ; ; ♂ w + Nsl1R::GFP [w +m] Y + TM6B w + GAL4 mat α-tub [w+m] Nsl1R::GFP [w +m] w + Df(3R)BSC318 ; ; ♂ x ; ; ♀ ♀ Y + TM6B w + TM6C w + GAL4 mat α-tub [w+m] Nsl1R::GFP [w +m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6C expression of markers confirmed by microscopy

Recombination of multiple fluorescent markers and GAL4 mat α-tub (V37) driver on the third chromosome w + GAL4 mat α-tub [w+m] GFP-CID [w+m] w + H2Av::mRFP [w +m] ; ; ♀ ♀ x ; ; ♂ ♂ w + TM6C Y + H2Av::mRFP [w +m] w w + x + + GAL4 mat α-tub [w+m] GFP-CID [w+m] ♀ ♀ ; ; ♂ ♂ ; ; Y w + H2Av::mRFP [w +m] + + w + GAL4 mat α-tub [w+m] GFP-CID [w+m] H2Av::mRFP [w+m] w + msps ; ; x ♂ ; ; Y + + Y + TM6C ♀ ♀ w + GAL4 mat α-tub [w+m] GFP-CID [w+m] H2Av::mRFP [w+m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6C w + GAL4 mat α-tub [w+m] GFP-Rod [w+m] w + H2Av::mRFP [w +m] ; ; x ; ; ♂ ♂ w + TM6C ♀ ♀ Y + H2Av::mRFP [w +m] w + GAL4 mat α-tub [w+m] GFP-Rod [w+m] w + + ; ; ♀ ♀ x ; ; ♂ ♂ w + H2AV::mRFP [w +m] Y + + w + GAL4 mat α-tub [w+m] GFP-Rod [w+m] H2Av::mRFP [w+m] w + msps ; ; ♂ x w ; ; Y + + TM6C ♀ ♀ + w + GAL4 mat α-tub [w+m] GFP-Rod [w+m] H2Av::mRFP [w+m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6C w + GAL4 mat α-tub [w+m] Nsl1R::GFP [w +m] w + H2Av::mRFP [w +m] ; ; ♀ ♀ x ; ; ♂ ♂ w + TM6C Y + H2Av::mRFP [w +m] w + GAL4 mat α-tub [w+m] Nsl1R::GFP [w +m] w + + ; ; ♀ ♀ x ; ; ♂ ♂ w + H2AV::mRFP [w +m] Y + + w + GAL4 mat α-tub [w+m] Rod::GFP [w+m] H2Av::mRFP [w+m] w + msps ; ; ♂ x ; ; Y + + w + TM6C ♀ ♀ w + GAL4 mat α-tub [w+m] Rod::GFP [w+m] H2Av::mRFP [w+m] ; ; TM6C ♀ ♀ ♂ ♂ Y/w + expression of markers confirmed by microscopy

(compound chromosome)
Combining FRT cal1 mutation with fluorescent markers w + FRT nanos GAL4 [w+m] cal1 w Sco MKRS ; ; ♀ ♀ x ; ; w + TM6B Y Cyo TM6B ♂ ♂ w + FRT nanos GAL4 [w+m] cal1 ; ; ♂ ♂ Y Cyo TM6B w Mis12::GFP [w +m] RCC1::mCherry [w+m] + w Ap ; ; ♂ ♂ x ; ♀ ♀ Y Cyo + Y Cyo ; TM3 (compound chromosome) w Mis12::GFP [w +m] RCC1::mCherry [w+m] + ; ; ♀ ♀ w Cyo TM3 w Mis12::GFP [w +m] RCC1::mCherry [w+m] + w + FRT GAL4 [w+m] cal1 ; ; ♀ ♀ x ; w Cyo TM3 Y Cyo ; ♂ ♂ TM6B w Mis12::GFP [w +m] RCC1::mCherry [w+m] FRT GAL4 [w+m] cal1 ; ; ♀ ♀ ♂ ♂ Y/w Cyo TM3

Combining FRT cenp-meta mutation with fluorescent markers w c-met FRT + w Sco MKRS ; ; ♀ ♀ x ; ; w Cyo + Y Cyo TM6B ♂ ♂ w c-met FRT + ; ; ♂ ♂ Y Sco TM6B w Ap w + nanos GAL4 RCC1::mCherry Mis12::GFP ♂ ♂ x ; ♀ ♀ ; ; w Cyo ; TM3 Y + TM6B (compound chromosome) w + nanos GAL4 RCC1::mCherry Mis12::GFP ; ; ♀ ♀ w Cyo TM3, Sb w + nanos GAL4 RCC1::mCherry Mis12::GFP w c-met FRT + ; ; ♀ ♀ x ; ; ♂ ♂ w Cyo TM3, Sb Y Sco TM6B w c-met FRT nanos GAL4 RCC1::mCherry Mis12::GFP ; ; ♀ ♀ ♂ ♂ Y/w Cyo TM6B

Combining incenp mutation with fluorescent markers + incenp + w Sco MKRS ; ; ♀ ♀ x ; ; + Cyo + Y Cyo TM6B ♂ ♂ + incenp + ; ; Y Sco TM6B ♂ ♂ w + nanos GAL4 RCC1::mCherry Mis12::GFP w Ap ; ; ♀ ♀ x ; w + Y Cyo ♂ ♂ TM6B ; TM3 (compound chromosome) w + nanos GAL4 RCC1::mCherry Mis12::GFP ; ; ♀ ♀ w Cyo TM3, Sb w + nanos GAL4 RCC1::mCherry Mis12::GFP + incenp + ; ; ♀ ♀ x ; ; w Cyo TM3, Sb Y Sco TM6B ♂ ♂ +/w incenp nanos GAL4 RCC1::mCherry Mis12::GFP ; ; ♀ ♀ ♂ ♂ w/Y Cyo TM6B

Recombination of sentin mutation and fluorescent markers on the third chromosome
nanos GAL4 Mis12::GFP sentin Cen 3 RCC1:: mCherry w + nanos GAL4 [w+m] RCC1::mCherry [w +m] Mis12::GFP [w+m] w + sentin ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6B w + TM6C w + nanos GAL4 [w+m] RCC1::mCherry [w +m] Mis12::GFP [w+m] w + + x ; ; ♀ ♀ ; ; ♂ ♂ w + sentin Y + + w + nanos GAL4 [w+m] RCC1::mCherry [w +m] Mis12::GFP [w+m] w + msps x ; ; ♂ ; ; ♀ ♀ Y + + w + TM6C w + nanos GAL4 [w+m] RCC1::mCherry [w +m] Mis12::GFP [w+m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6C expression of markers confirmed by microscopy w + nanos GAL4 [w+m] RCC1::mChrry [w +m] Mis12::GFP [w+m] w + sentin ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6C w + TM6C w + nanos GAL4 [w+m] RCC1::mCherry [w +m] Mis12::GFP [w+m] ; ; ♀ ♀ w + sentin sterile = sentin mutation present

Recombination of sentin mutation and fluorescent markers on the third chromosome
nanos GAL4 sentin Cen 3 GFP-Rod (unknown location) RCC1:: mCherry w + nanos GAL4 [w+m] RCC1::mCherry [w +m] GFP-Rod [w+m] w + sentin ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6B w + TM6C w + nanos GAL4 [w+m] RCC1::mCherry [w +m] GFP-Rod [w+m] w + + ; ; ♀ ♀ x ; ; ♂ ♂ w + sentin Y + + w + nanos GAL4 [w+m] RCC1::mCherry [w +m] GFP-Rod [w+m] w + msps ; ; ♂ x ; ; ♀ ♀ Y + + w + TM6C w + nanos GAL4 [w+m] RCC1::mCherry [w +m] GFP-Rod [w+m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6C expression of markers confirmed by microscopy w + nanos GAL4 [w+m] RCC1::mChrry [w +m] GFP-Rod [w+m] w + sentin ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6C w + TM6C w + nanos GAL4 [w+m] RCC1::mCherry [w +m] GFP-Rod [w+m] w ; ; ♀ ♀ + sentin sterile = sentin mutation present

Recombination of nsl1R mutation and FRT sequence on the first chromosome
FLP kmn1 FRT 9-2 Cen 1 w f w kmn1 [w +m] + + w f hsFLP FRT [w +m] + + ; ; ♀ ♀ x ; ; ♂ ♂ FM7C + + Y + + w kmn1 [w +m] + + FMO w f [w +m] + + ; ; ♀ ♀ x ; ; ♂ ♂ w f hsFLP FRT [w+m] + + Y + + w kmn1 f hsFLP FRT [w +m] + + FMO w f [w +m] + + ; ; ♀ x ; ; ♂ ♂ FMO w f [w +m] + + Y + + w kmn1 f hsFLP FRT [w +m] + + ; ; ♀ ♀ ♂ ♂ FMO w f [w +m] + + Recombination of mis12 mutation and FRT sequence on the third chromosome mis12 FRT 2A Cen 3 ru h th st w + mis12 [w +m] w + ru h th st FRT [w+m] ; ; ♀ ♀ x ; ; ♂ ♂ w + TM6B Y + ru h th st FRT [w+m] w + mis12 [w +m] w + ru h th st Pr ; ; ♀ ♀ ; ; ♂ ♂ w + ru h th st FRT [w+m] x Y + TM6B w + mis12 th st FRT [w +m] w + ru h th st Pr ; ; ♂ x ; ; ♀ ♀ Y + ru h th st Pr w + TM6B w + mis12 th st FRT [w +m] ; ; ♀ ♀ ♂ ♂ w/Y + TM6B

Recombination of cal1 mutation and FRT sequence on the third chromosome
FRT 82B Cen 3 neo cal1 cu sr e ca w + cal1 + + FRT cu sr es ca ; ; ♀ ♀ x ; ; ♂ ♂ w + TM6C Y + FRT cu sr e ca w + cal1 w + Pr cu sr es ca ; ; ♀ ♀ x ; ; ♂ ♂ + + FRT cu sr es ca Y + TM6B w + FRT cu cal1 w + Pr cu sr e ca ; ; ♂ x ; ; ♀ ♀ Y + Pr cu sr es ca w + TM6B w + FRT cu cal1 ; ; ♀ ♀ w/Y + ♂ ♂ TM6B Recombination of cenp-meta mutation and FRT sequence on the second chromosome FRT 40A c-met neo Cen 2 al dp b pr + al dp b pr Bl + w al dp b pr FRT + ; ; ♀ ♀ x ; ; ♂ ♂ + Cyo + Y ru h th st FRT + + al dp b pr FRT + w c-met + ; ; ♂ ♂ x ; ; ♀ ♀ Y Cyo + w Cyo + w c-met + + al dp b pr Bl + ; ; ♀ ♀ ; ; ♂ ♂ + al dp b pr FRT + x Y Cyo + w c-met pr FRT + w Sco + ; ; ♂ x ; ; ♀ ♀ Y al dp b pr Bl + w Cyo + w c-met pr FRT + ; ; ♀ ♀ ♂ ♂ Y Cyo +

Recombination of FRT cal1 sequence with GFP-Rod on the third chromosome
FRT 82B Cen 3 neo cal1 cu e w + FRT cu cal1 w + e GFP-Rod [w+m] ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6B w + e GFP-Rod [w+m] w + FRT cu cal1 w + Pr cu e ; ; ♀ ♀ x ; ; ♂ ♂ w + e GFP-Rod [w+m] Y + TM6B w + FRT cu cal1 GFP-Rod [w+m] w + msps ; ; x w ; ; ♀ ♀ Y + Pr cu e ♂ + TM6C w + FRT cu cal1 GFP-Rod [w+m] ; ; ♀ ♀ ♂ ♂ Y/w + TM6C w + FRT cu cal1 GFP-Rod [w+m] w + cal1 ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6C w + TM6C w + FRT cu cal1 GFP-Rod [w+m] ; ; ♂ ♂ Y + cal1 lethal = cal1 mutation present

Recombination of FRT sequence and nanos Gal4 on the third chromosome
FRT 82B nanos GAL4 Cen 3 neo cu cal1 w + FRT cu cal1 w + GAL4 [w+m] ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6B w + TM6B w + FRT cu cal1 w + Pr cu ; ; ♀ ♀ x ; ; ♂ ♂ Y + GAL4 [w+m] Y + TM6B w + FRT GAL4 [w+m] w + Pr cu ; ; ♂ x ; ; ♀ ♀ Y + Pr cu w + TM6B w + FRT GAL4 [w+m] ; ; ♀ ♀ ♂ ♂ w/Y + TM6B Recombination of FRT nanos Gal4 and cal1 mutation on the third chromosome FRT 82B nanos GAL4 Cen 3 neo cu cal1 w + FRT cu cal1 w + FRT GAL4 [w+m] ; ; ♀ ♀ x ; ; w + TM6B Y + TM6B ♂ ♂ w + FRT cu cal1 w + Pr cu ; ; x w ♀ ♀ ; ; ♂ ♂ + FRT GAL4 [w+m] Y + TM6B w + FRT GAL4 [w+m] cal1 x w + Df(3R)Exel6176 ; ; ♂ Y ; ; ♀ ♀ + Pr cu w + TM6B w + FRT GAL4 [w+m] cal1 w + FRT GAL4 [w+m] cal1 ; ; ♀ ♀ ♂ ♂ ; ; w + TM6B w + Df(3R)Exel6176 lethal = cal1 mutation present

Recombination of sentin and wac mutants on the third chromosome
+ wac w + sentin ; ; ♀ ♀ x ; ; w + TM6C ♂ ♂ Y + TM6C w + wac w + Pr ; ; ♀ ♀ x ; ; ♂ ♂ w + sentin Y + TM6B w + rec (wac sentin])? w + Df(3L)ED4515 ; ; ♂ ; ; ♀ ♀ Y + Pr x w + TM6C w + rec (wac sentin)? w + rec (wac sentin)? ; ; ♂ ♂ ; ; 15♀ Y + TM6C w + Df(3L)ED4515 sterile = sentin mutation present w + rec (wac sentin)? w + wac ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6C w + TM6C w + rec (wac sentin)? w ; ; ♀ ♀ + wac sterile = wac mutation present w + wac sentin w + Df(3L)ED4515 ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM6C w + TM6C w + wac sentin ; ; ♀ ♀ ♂ ♂ Y/w + TM6C

Induction of kmn1 mutation in germline clone
w kmn1 hsFLP FRT9-2 [w +m] + + w ovoD FRT9-2 [w +m] + + ; ; ♀ ♀ x ; ; ♂ ♂ FMO w [w +m] + + Y + + hs w kmn1 hsFLP FRT9-2 [w +m] + + ; ; ♀ ♀ w ovoD FRT9-2 [w +m] + + Induction of mis12 mutation in germline clone w + ovoD FRT2A hsFLP w + Dr ; ; ♂ ♂ x ; ; Y + ♀ ♀ + w + TM3 hsFLP w + Dr w + mis12 FRT2A ; ; ; ; ♀ ♀ Y + ovoD FRT2A ♂ ♂ x w + TM6B hs hsFLP w + mis12 FRT2A ; ; ♀ ♀ w + ovoD FRT2A Induction of nuf2 mutation in germline clone hsFLP w ovoD FRT40A + x w ; nuf2 FRT40A + ; ; Cyo ; ♀ ♀ Y Cyo + ♂ ♂ w + hsFLP w nuf2 FRT40A + ; ; ♀ ♀ w ovoD FRT40A + Induction of cenp-meta mutation in germline clone hsFLP w ovoD FRT40A + x w c-met FRT40A + ; ; ♂ ♂ ; ; ♀ ♀ Y Cyo + w Cyo + hsFLP w c-met FRT40A + ; ; w ovoD FRT40A + ♀ ♀

Induction of cal1 mutation in germline clone (with GFP-Rod on the third chromosome; location not known) w + ovoD FRT82B hsFLP w + Dr ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM3 w + TM3 hsFLP w + Dr w + FRT cu cal1 (GFP-Rod) ; ; x Y + ovoD FRT82B ♂ ♂ ; ; ♀ ♀ w + TM6B hs hsFLP w + FRT cu cal1 (GFP-Rod) ; ; ♀ ♀ w + ovoD FRT82B Induction of cal1 mutation in germline clone (with fluorescent markers on the second chromosome) w + ovoD FRT82B hsFLP w + Dr ; ; ♂ ♂ x ; ; ♀ ♀ Y + TM3 w + TM3 hsFLP w + Dr w RCC1::mCherry Mis12::GFP FRT cu cal1 GAL4 ; ; x ; ; ♀ ♀ Y + ovoD FRT82B ♂ ♂ w Cyo TM3 hs hsFLP w RCC1::mCherry Mis12::GFP FRT cu cal1 nanos GAL4 w ; ; ♀ ♀ + ovoD FRT82B

A transgene 1 2 3 B PCR fly line AD1 AD2 AD3 AD4 AD5 AD6 DLIC::GFP ln1 ns 2L(29D1) ns ND ND ND DLIC::GFP ln2 ns 3L(79A2) ns ND ND ND TAIL-2 DLIC::GFP ln3 ns X(8F9) ns ND ND ND DLIC::GFP ln4 ns 3L(79A2) ns ND ND ND DLIC::GFP ln1 ns ns ns ND ND ND DLIC::GFP ln2 - 3L(79A2) ns ND ND ND TAIL-3 DLIC::GFP ln3 ns - ns ND ND ND DLIC::GFP ln4 3L(79A2) 3L(79A2) ns ND ND ND Mis12::GFP ln1 ND ND ND 3L(64B13) ns ns Mis12::GFP ln2 - - ND ND ND 2R(53F8) TAIL-2 Mis12::GFP ln3 ND ND ND - - ns Mis12::GFP ln4 - ns ND ND ND - ns Mis12::GFP ln1 ND ND ND 3L(64B13) ns Mis12::GFP ln2 ND ND ND - - 2R(53F8) TAIL-3 Mis12::GFP ln3 - ND ND ND - ns Mis12::GFP ln4 ND ND ND X(9E1) - ns Fig.8. Tab. TAIL-PCR successfully maps the position of transgenes in Drosophila genome. A Representation of the P element (light blue) insertion in the genome (dark blue). The black arrows symbolize arbitrary degenerate (AD) primers, which anneal randomly throughout the genome. Red arrows symbolize primers specific for 3’ end of P element 5’-directed. Primers 1, 2 and 3 are used in the three consecutive rounds of PCR. A product generated from a nested primer and an arbitrary primer is specific. B Results of TAIL-PCR mapping. Products of TAIL-PCR were run on an agarose gel. Products from TAIL-2 and TAIL-3, that gave rise to defined bands were sequenced. Sequences were then submitted to BLAST and the results are presented in the table (ns, not sequenced). Ln, fly line representing individual insertion site; R, right arm of chromosome; L, left arm of chromosome; -, unsuccessful mapping. ND, not determined.

-10 min -3 min 0 min 7 min 9 min GFP::α-Tubulin RCC1::mCherry merge 11 min 13 min 15 min 17 min 21 min GFP::α-Tubulin RCC1::mCherry merge merge Fig.9. The bipolar spindle is assembled around 20 min after initiation of abrupt chromosome movement at the nuclear envelope breakdown (NEB). Spindle formation was filmed in oocytes from nanos GAL4 UASp GFP::α-Tubulin RCC1::mCherry heterozygotes maturated for 5 days at 18°C. 3 min after the signal of GFP::α-Tubulin is of the same intensity in and out of the nucleus area, the chromosomes initiate abrupt movement towards dorsal side of the oocyte (t0, orange area). 7 min after that, signal of GFP::α-Tubulin increases around chromosomes. Microtubules assemble around chromosomes and elongate. At 21 min, the bipolar shape of the spindle is visible. This film was chosen as it is representative for the timing of events after NEB. Data based on 5 oocytes, in which formation of a bipolar spindle was observed. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Z sections were cropped individually for every time point shown. Contrast was adjusted for full projection of individual image. Scale 10 μm.

GFP RCC1::mCherry merge α-Tubulin Subito and Tubulin Incenp Deterin Fig.10. GFP::Subito, Incenp::GFP and Deterin::GFP mark the central spindle. Division apparatus in oocytes from the following transheterozygotes maturated for 5 days at 18°C was observed: nanos GAL4 UASp GFP::α-Tubulin RCC1::mCherry, nanos GAL4 UASp GFP::α-Tubulin GFP::Subito, nanos GAL4 RCC1::mCherry Incenp::GFP and nanos GAL4 RCC1::mCherry Deterin::GFP. Fluorescent signal of Subito, Incenp and Deterin was observed adjacent to chromosomes. GFP::Subito significantly marks the central spindle (short line) when co-expressed with GFP::α-Tubulin used to visualize the whole spindle (long line). In addition to the central spindle, Incenp::GFP and Deterin::GFP localize to centromeres (arrowheads). Incenp is also seen as a thread joining homologs of the fourth chromosome (arrow). Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

5 min 14 min 16 min 17 min Incenp:GFP RCC1::mCherry merge 24 min 33 min 36 min 54 min -1 min +1 min Incenp:GFP RCC1::mCherry merge Fig.11. Incenp::GFP dynamically changes location in the division apparatus during spindle formation. Figure description of the following page.

Fig.11. Incenp::GFP dynamically changes location in the division apparatus during spindle formation. Incenp::GFP behaviour during spindle formation was filmed in oocytes from nanos GAL4 UASp RCC1::mCherry Incenp::GFP heterozygotes, maturated 5 days at 18°C. Incenp::GFP localization was followed after its initial localization (t0), co-incident with chromosome movement accompanying RCC1::mCherry signal diffusion from nuclear area. Incenp::GFP initially localizes to chromosomes (narrow arrow) and concentrates in a distinct chromatin region. From that region, it transfers onto presumptive centromeres (arrowheads) and edges of the chromosomes (thick arrow), likely corresponding to the forming central spindle. Data based on analysis of 2 oocytes until around 25 min after the initiation of the abrupt chromosome movement and on 1 oocyte until Incenp::GFP behaviour is static. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Z sections were cropped individually for every time point shown. Contrast was adjusted for full projection of the whole film. Scale 5 μm.

throughout the spindle
GFP::Klp61F RCC1::mCherry merge A toward the poles throughout the spindle central spindle B STAGE early 13 late 13 14 Spindle length in GFP::Klp61F localization patterns [μm] Spindle length in oocyte stages based on GFP::α-Tubulin signal [μm] early 13 (n=6) late 13 n=(10) 14 (n=27) toward the poles (n=16) throughout the spindle (n=17) central spindle (n=22) STAGE Fig.12. GFP::Klp61F is a marker of metaphase spindle maturation. Figure description of the following page.

Fig. 12. GFP::Klp61F is a marker of metaphase spindle maturation
Fig.12. GFP::Klp61F is a marker of metaphase spindle maturation. GFP::Klp61F localization was analysed in oocytes from nanos GAL4 UASp RCC1::mCherry GFP::Klp61F heterozygotes, maturated 5 days at 18°C. A GFP::Klp61F displays 3 types of localization on the spindle. In the first one, GFP::Klp61F localizes stronger towards the poles, but weaker at the very poles. In the second type, GFP::Klp61F is distributed throughout the spindle. In the third type, GFP::Klp61F is concentrated at the central spindle, but excluded from the very middle of the central spindle (arrow). B GFP::Klp61F localization pattern is correlated with the spindle length. This is likely associated with oocyte stage, as oocytes in early stages after NEB are longer than in later oocyte stages, measured in oocytes from nanos GAL4 UASp GFP::α-Tubulin RCC1::mCherry heterozygotes. Therefore, different localization pattern of GFP::Klp61F may represent different stage of the spindle maturation after establishment of bipolarity. Staging was based on dorsal appendages and nurse cells content. Data based on a single cross to generate GFP::α-Tubulin expressing flies and three crosses to generate GFP::Klp61F expressing flies. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm. RCC1::mCherry -5 min 0 min 24 min 29 min 44 min GFP::Klp61F merge Fig.13. GFP::Klp61F is localized towards the poles just after the spindle is formed. GFP::Klp61F behaviour during spindle formation was filmed in oocytes from nanos GAL4 UASp RCC1::mCherry GFP::Klp61F heterozygotes, maturated 5 days at 18°C. GFP::Klp61F localization towards the poles is visible around 25 min after initiation of the abrupt chromosome movement (t0), the time of the formation the bipolar spindle observed earlier. Data based on analysis of a single oocyte, in which formation of a bipolar spindle was observed. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Z sections were cropped individually for every time point shown. Contrast was adjusted for full projection of individual image. Scale 10 μm. GFP::Mei-38 RCC1::mCherry merge Fig.14. GFP::Mei-38 displays strong localization towards spindle poles. GFP::Mei-38 localization was analysed in oocytes from nanos GAL4 UASp RCC1::mCherry GFP::Mei-38 heterozygotes, maturated 5 days at 18°C. Z sections for the images were taken at 0.5 μm step size. Scale bar 10um

A Lamin Dm0::GFP RCC1::mCherry merge B RCC1::mCherry -5 min 0 min 5 min 15 min 60 min Lamin Dm0::GFP merge Fig.15. Lamin Dm0::GFP disperses uniformly after nuclear envelope breakdown (NEB). Lamin Dm0::GFP localization and behaviour during spindle formation was analysed in oocytes from nanos GAL4 UASp RCC1::mCherry Lamin Dm0::GFP heterozygotes, maturated 5 days at 18°C. A No concentration of Lamin Dm0::GFP fluorescence in the area of chromosomes was observed in mature oocytes. Z sections for the images were taken at 0.5 μm step size. B Lamin Dm0::GFP disperses from the nuclear area after initiation of the abrupt chromosome movement (t0). The arrow shows the edge of the nucleus with Lamin:Dm0 inside the nucleus. Data based on observation of a single oocyte. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Scale 10 μm.

A GFP::hUtrCH RCC1::mCherry merge B 0 min 5 min 6 min 8 min 10 min GFP::hUtrCH RCC1::mCherry merge Fig.16. GFP::hUtrCH is recruited to the area around and within the chromatin mass after nuclear envelope breakdown (NEB). GFP::hUtrCH localization and behaviour during spindle formation was filmed in oocytes from nanos Gal4 UASp RCC1::mCherry GFP::hUtrCH transheterozygotes, maturated 5 days at 18°C. A Fluorescent signal of hUtrCH (shape of the dotted line) in mature oocytes was observed at distance from the chromosomes, in a shape resembling the spindle. Z sections for the images were taken at 0.5 μm step size. B GFP::hUtrCH localizes to chromosomes around 5 min after initiation of the abrupt chromosome movement (t0). With time, the signal becomes stronger and is visible at the chromosome area and around. Data based on analysis of a single oocyte. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Z sections were cropped individually for every time point shown. Contrast was adjusted for full projection of individual image. Scale 10 μm.

A Axs::GFP RCC1::mCherry merge B 0 min 5 min 6 min 7 min 10 min Axs::GFP RCC1::mCherry merge Fig.17. Axs::GFP is recruited to the area around chromatin mass after nuclear envelope breakdown (NEB). Axs::GFP localization in mature oocytes and behaviour during spindle formation was filmed in oocytes from nanos GAL4 UASp RCC1::mCherry Axs::GFP heterozygotes, maturated 5 days at 18°C. A Fluorescent signal of Axs was observed at distance from the chromosomes, in a shape resembling the spindle. Z sections for the images were taken at 0.5 μm step size. B Axs::GFP signal is visible in the area of chromosomes around 5 min after initiation of the abrupt chromosome movement (t0). It seems to surround chromatin mass. Data based on analysis of a single oocyte. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Z sections were cropped individually for every time point shown. Contrast was adjusted for full projection of individual image. Scale 10 μm.

chromosomes within the spindle
GFP::RCC1 RCC1::mCherry merge karyosome chromosomes within the spindle Fig.18. GFP::RCC1 marks the karyosome and chromosomes in mature oocytes. GFP::RCC1 localization was analysed in oocytes from nanos GAL4 UASp RCC1::mCherry GFP::RCC1 transheterozygotes, maturated 5 days at 18°C. Z sections for the images were taken at 0.5 μm step size. Scale 5 μm. Nod::GFP RCC1::mCherry merge karyosome chromosomes within the spindle Fig.19. Nod::GFP marks the karyosome but not chromosomes in mature oocytes. GFP::RCC1 localization was analysed in oocytes from nanos GAL4 UASp RCC1::mCherry GFP::RCC1 transheterozygotes, maturated 5 days at 18°C. Intensity of Nod::GFP signal corresponds density of chromatin marked by RCC1::mCherry, except one strong focus. The arrow shows the edge of the nucleus with Lamin::Dm0 inside the nucleus. Z sections for the images were taken at 0.5 μm step size. Scale 5 μm.

A -30 min -20 min -10 min 0 min 8 min Nod::GFP RCC1::mCherry merge B 4 min 7 min 10 min 20 min 30 min Nod::GFP RCC1::mCherry merge Fig.20. Nod::GFP signal peaks at the nuclear envelope breakdown (NEB) and diminishes during spindle formation. Nod::GFP behaviour before and during the time of spindle formation was filmed in oocytes from nanos GAL4 UASp RCC1::mCherry Nod::GFP heterozygotes, maturated 5 days at 18°C. A Fluorescent signal of Nod was observed on chromosomes before NEB and dramatically increases at the time of NEB. The signal peaks 8 min after the abrupt chromosome movement (t0). B Nod::GFP signal raises after the initiation of the abrupt chromosome movement and peaks around 4 min after that moment (t0). Nod::GFP signal decreases and around 30 min from t0 it is not visible in the area of chromosomes with the same imaging settings. Data based on analysis of a single oocyte before and after NEB each. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Scale 10 μm.

chromosomes within the spindle
A SuVar(205)::GFP RCC1::mCherry merge karyosome chromosomes within the spindle B RCC1::mCherry -70 min t0 10 min 15 min 20 min SuVar(205)::GFP merge Fig.21. Su(var)205::GFP signal is dramatically reduced from chromatin after nuclear envelope breakdown (NEB). Su(var)205::GFP localization and behaviour after NEB was filmed in oocytes from heterozygotes of nanos GAL4 UASp RCC1::mCherry co-expressing Su(var)205::GFP under its own promoter, maturated 5 days at 18°C. A Su(var)205::GFP localizes to an area within karyosome before NEB. In mature oocytes, Su(var)205::GFP is localized to an expected pericentric heterochromatin. Z sections for the images were taken at 0.5 μm step size. B Su(var)205::GFP behaviour was followed after nuclear envelope breakdown, marked by RCC1::mCherry signal diffusion from nuclear area. Before NEB (t0), Su(var)205::GFP signal is strong on chromosomes after NEB, Su(var)205::GFP signal rapidly decreases until it is not visible with the same imaging settings. Data based on analysis of a single oocyte. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Scale 5 μm.

A GFP RCC1::mCherry merge Mis12 Nsl1R CID B -9 min 0 min 7 min 8 min 9 min 12 min GFP-CID RCC1::mCh merge Fig.22. Mis12, Nsl1R and CID mark the position of kinetochores/centromeres on chromatin. Chromosomes in oocytes from the following heterozygotes maturated for around 5 days at 18°C was observed: nanos GAL4 UASp Mis12::GFP RCC1::mCherry, nanos GAL4 UASp Nsl1R::GFP RCC1::mCherry and nanos GAL4 RCC1::mCherry co-expressed with GFP-CID (expressed under its own promoter). A Fluorescent foci of Mis12, Nsl1R and CID in mature oocytes were observed at the edges of chromosome, along their long axis. Z sections for the images were taken at 0.5 μm step size. B GFP-CID forms clusters of signal within karyosome. Around 8 min after initiation of abrupt chromosome movement GFP-CID foci decluster and change position within chromatin mass. Data based on analysis of a single oocyte. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Contrast was adjusted for full projection of individual image. Scale 5 μm.

GFP::α-tubulin Mis12::mCherry merge Fig.23. Mis12::mCherry and GFP::α-tubulin show the relative position of kinetochores and microtubules. Mis12::GFP localization was observed in oocytes of Mis12::mCherry GFP::α-tubulin maternal α-tubulin (V37) heterozygotes, maturated 3 days at 25°C. Mis12::mCherry is located at the edge of chromosome exclusion area, adjacent to microtubules. Z sections for the images were taken at 0.5 μm step size. Binning 2. Scale 10 μm. 12 min 20 min 31 min 51 min 54 min 55 min Fig.24. DLIC::GFP signal fluctuates on division apparatus during chromosome positioning. DLIC::GFP behaviour during spindle assembly was observed in oocytes from DLIC::GFP RCC1::mCherry nanos GAL4 heterozygotes, maturated 5 days at 18°C. DLIC::GFP localizes to chromosomes 12 min after initiation of abrupt chromosome movement (arrowhead). At 20 min DLIC::GFP signal is visible thoughout the spindle As chromosomes oscillate, stronger DLIC::GFP signal is present close to chromosomes. Intensity of DLIC::GFP is uniform after chromosomes stop oscillations. Data based on analysis of a single oocyte. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Z sections were cropped individually for every time point shown. Contrast was adjusted for full projection of individual image. Scale 10 μm.

A GFP-Rod t0 40 sec 80 sec 120 sec 160 sec B GFP-Rod -10 min -8.5 min -8 min -6.5 min -6 min -4 min -2.5 min -2 min -0.5 min t0 GFP-Rod Mis12::mCherry merge Fig.25. GFP-Rod streams from kinetochores attached by microtubules in mature spindles. A GFP-Rod behaviour was observed in oocytes from GFP-Rod RCC1::mCherry nanos GAL4 heterozygotes, maturated 5 days at 18°C (GFP-Rod expressed under its own promoter). GFP-Rod streams from chromosome area towards the spindle poles (arrow and arrowhead). Z sections for the images were taken at 0.5 μm step size, filmed 40 sec/min. B GFP-Rod behaviour was observed in oocytes from GFP-Rod Mis12::mCherry nanos GAL4 heterozygotes, maturated 3 days at 25°C. GFP-Rod signal initiates at 8 distinct foci and then transfers on microtubules (colour-coded foci are 2x magnified in the insets). Exposure of Mis12::mCherry to 100% laser power after 10 min observation of GFP-Rod behaviour confirms that the foci represent individual kinetochores (below). Data based on 3 oocytes from a single cross to obtain the heterozygotes. Z sections for the images were taken at 0.5 μm step size, filmed 2 time point/min. Scale 10 μm.

NEB HP1 CID centromere de-clustering; kinetochore assembly microtubule nucleation spindle envelope formation HP1 Nod ! ! Actin Axs CPC Mis12 Nsl1R ! central spindle formation chromosome re-arrangements Nod CPC Mis12 Nsl1R ! Subito CPC Actin Axs spindle focusing stable kinetochore-microtubule attachment ! Mei-38 Klp61F CPC Mis12 Nsl1R Subito CPC Rod Dynein ! Actin Axs spindle shortening Mei-38 CPC Mis12 Nsl1R ! Klp61F Subito CPC Rod Dynein Actin Axs Fig.26. Model for timely localization of new markers of the division apparatus in oocytes. Figure description of the following page.

Fig.26. Model for timely localization of new markers of the division apparatus in oocytes. Spindle (green) formation initiates after nuclear envelope breakdown (NEB). Spindle microtubules nucleate around chromosomes, elongate and become focused. Bipolar focused spindle shortens over time. The processes of spindle formation and maturation are accompanied by dynamic behaviour of markers for different regions of the spindle (green) and chromosomes (red and blue), as well as spindle-associated structures such as the spindle envelope (yellow). Exclamation mark shows the estimated peak of localization a marker on the division apparatus in the area indicated by an arrow.

RCC1::mCherry t0 4 min 14 min 15 min 16 min 17 min Mis12::GFP merge 19 min 20 min 21 min 22 min 23 min 24 min Mis12::GFP merge 36 min 37 min 38 min 39 min 40 min 41 min Mis12::GFP merge 51 min 54 min 61 min 64 min 67 min 72 min merge Fig.27. Chromosome positioning in Drosophila oocytes is kinetochore-led. Figure description of the following page.

Fig.27. Chromosome positioning in Drosophila oocytes is kinetochore-led. Kinetochore and chromosome behaviour was analysed in oocytes from nanos GAL4 UASp RCC1::mCherry Mis12::GFP heterozygotes, maturated 5 days at 18°C. First panel: Mis12::GFP foci start to be visible around 4 min after initiation of an abrupt chromosome movement (arrowhead). Kinetochores lead chromatin protrusions from the main chromatin mass at around 15 min. Chromatin protrusions appear on the axis of the following chromosome movement (two-sided arrow). Second panel: kinetochores lead chromosome orientation change. Black lines help to see position of a kinetochore and chromosome arm in time of the chromosome changing orientation. Third panel: kinetochores lead separation of bi-oriented chromosomes from remaining chromatin mass. Fourth panel: bi-oriented chromosomes change relative position within the chromatin mass and oscillate once congressed. Data on chromosome behaviour based on 3 oocytes in which congression and bipolarity of chromosomes where achieved during the filming time. Oocytes derived from 3 individual crosses to generate heterozygotes co-expressing fluorescently marked kinetochores (either Mis12 or Nsl1R) and RCC1::mCherry, expression driven by nanos GAL4. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Images were cropped in XY at fixed position, for each panel individually. Contrast was adjusted for full projection of the whole film. Scale 5 μm.

RCC1::mCherry t0 11 min 13 min 14 min 18 min GFP-Rod merge 28 min 29 min 30 min 31 min 32 min GFP-Rod merge 42 min 49 min 65 min 73 min 82 min GFP-Rod merge Fig.28. Weak kinetochore-microtubule attachments lead chromosome movements. Kinetochore-microtubule attachment was analysed in oocytes from nanos GAL4 UASp RCC1::mCherry co-expressing GFP-Rod under its own promoter, maturated 5 days at 18°C. First panel: GFP-Rod starts to localize at chromosomes (t0; arrow) and accumulates as a single focus, followed by de-clustering and occasional stream events (arrowheads). Second panel: Chromosome change of orientation is not accompanied by GFP-Rod stream. Third panel: Chromosome congression occurs with GFP-Rod frequently accumulated at kinetochores. Data on GFP-Rod behaviour from nuclear envelope breakdown until chromosome congression is based on 2 oocytes. Timing of events varied insignificantly between the oocytes. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Contrast was adjusted for full projection of the fragment of film presented in each panel, separately. Scale 10 μm.

adult maturation time [days] no. of females analysed
kmn1 FRT/ovoD FRT adult maturation time [days] no. of females analysed no. of females with developed oocytes developed oocytes in non-hs flies with ovoD hs larvae 3 >30 >30 yes mis12 FRT/ovoD FRT adult maturation time [days] no. of females analysed no. of females with developed oocytes ovoD Mis12+ flies with developed oocytes hs larvae 3 17 (rec1); 25 (rec2) 2, 3, 4, 5 collectively hs pupae ~5 (rec1) hs adults 3 3 (rec1) no nuf2 FRT/ovoD FRT adult maturation time [days] no. of females analysed no. of females with developed oocytes ovoD Nuf2+ flies with developed oocytes hs larvae 3 104 4 14 hs adults 5 53 no 6 16 cal1 FRT/ovoD FRT adult maturation time [days] no. of females analysed no. of females with developed oocytes ovoD Cal1+ flies with developed oocytes rec1 rec2 rec1 rec2 rec1 rec2 3 4 18 25 hs larvae 4 7 15 33 hs pupae 6 6 2 rec1 rec2 rec1 rec2 rec1 rec2 4 8 5 hs adults 5 5 43 15 37 10 no 6 6 6 16 3 2 Cenp-meta FRT/ovoD FRT adult maturation time [days] no. of females analysed no. of females with developed oocytes ovoD Kmn1+ flies with developed oocytes hs larvae 3 32 29 no 4 26 22 5 12 6 Fig.29. FRT/FLP site-directed recombination results in production of homozygous cal1 and cenp-meta oocytes. Figure description of the following page.

Fig.29. FRT/FLP site-directed recombination results in production of homozygous cal1 and cenp-meta oocytes. kmn1, mis12, nuf2, cal1 and cenp-meta heterozygotes were heat shocked (hs) at indicated developmental stage to induce the site-directed mutagenesis. Larvae were heat shocked 5, 6 and 7 days before eclosing. Pupae were heat shocked on the day of eclosing. Adults were heat shocked within 3-4 hrs after eclosing. Heat shocked females were examined for the presence of developed oocytes in the ovaries after indicated time of adult maturation at 25°C. Only oocytes with eliminated ovoD, and thus homozygous for the kinetochore mutation, can be produced. This is with one exception for kmn1 in which oocytes are produced even without hs. Hs-induced mutations of mis12, nuf2, cal1 and cenp-meta heterozygotes yielded cal1 (2 recombinants, rec) and cenp-meta homozygous oocytes. cal1 homozygous oocytes were produced most efficiently after heat shock of adult females and maturating them 5 days. cenp-meta homozygous oocytes were produced after heat shock of larvae. Maturation of eclosed females for 3 days resulted in the most efficient oocyte production.

% oocytes with 7-8 Mis12::GFP foci
A Mis12:GFP RCC1::mCherry merge % oocytes with 7-8 Mis12::GFP foci wt 100 80 60 cal1 40 20 wt (n=16) cal1 (n=7) B % oocytes with GFP-Rod signal % oocytes 100 100 80 80 60 60 40 40 20 20 wt (n=92) cal1 (n=108) wt (n=33) cal1 (n=26) wt cal1 random GFP-Rod signal GFP-Rod signal only at kinetochores GFP-Rod signal on microtubules GFP-Rod Fig.30. cal1 mutation results in partial dysfunction of kinetochores. FRT cal1 (rec1) flies were crossed with FRT ovoD flies and mutant oocytes were analysed 5 days after heat shock of eclosed progeny and maturating the females at 25°C. A UASp Mis12::GFP RCC1::mCherry/+; FRT cal1 nanos-GAL4 /FRT cal1 nanos-GAL4 (cal1) oocytes have fewer Mis12::GFP foci on chromosomes observed after exposure to 20-40% laser power as compared to 7-8 Mis12::GFP foci visible in wild type (wt) oocytes of stage 14 after exposure to 20% LP. Data obtained from a single cross for cal1 mutant and for wt, with a use of different fly line expressing Mis12::GFP B GFP-Rod (expressed under its own promoter) signal is found at similar frequency as locally concentrated in oocytes from FRT cal1 GFP-Rod/FRT cal1 (cal1) and in mature oocytes from GFP-Rod/GFP-Rod flies (wt) (left chart). GFP-Rod signal is found at similar frequency arranged non-randomly, in a way suggesting a spindle structure in these two strains (right chart). GFP-Rod signal is found at similar frequency in a microtubule-like arrangement in these two strains (image and right chart). Data for cal1 mutant and wt obtained from two crosses. Z sections for the images were taken at 0.5 μm step size, n value represents number of oocytes analysed. Scale 5 μm.

CONGRESSED CHROMOSOMES in cal1 mutant oocytes
% oocytes with correctly oriented congressed chromosomes (4+4 or 3+4 on each side) % oocytes with congressed chromosomes 100 100 80 80 60 60 40 40 20 20 wt (n=16) cal1 (n=7) wt (n=16) cal1 (n=3) B Mis12::GFP RCC1::mCherry merge CONGRESSED CHROMOSOMES in cal1 mutant oocytes UNCONGRESSED CHROMOSOMES in cal1 mutant oocytes chrom. a chrom. b chrom. c Mis12::GFP 0 min 10 min 11 min 12 min 13 min merge RCC1::mCherry Fig.31. cal1 mutation results in increased frequency of uncongressed chromosomes and mis-position of kinetochores on congressed chromosomes. Figure description on the following page.

Fig.31. cal1 mutation results in increased frequency of uncongressed chromosomes and mis-position of kinetochores on congressed chromosomes. UASp Mis12::GFP RCC1::mCherry/+; FRT cal1 nanos-GAL4 /FRT cal1 nanos-GAL4 (cal1) oocytes were analysed 5 days after maturating the females at 25°C. A Chromosomes in cal1 mutant oocytes have decreased frequency of congressed chromosomes (left chart). Congressed chromosomes defined by position of chiasmatic chromosomes next to each other. Congressed chromosomes with correct number of kinetochore foci (7 or 8) have kinetochores within chromatin mass or kinetochores number on each side is incorrect (right chart). B Examples of congressed (top panel) and uncongressed chromosomes (bottom panel; 3 chromosomes within a single oocytes marked a, b, c are distant from each other) in cal1 mutant oocytes. The example of an oocyte with uncongressed chromosomes shows that some kinetochore-microtubule attachment may be present in cal1 mutant oocytes as chromosome c changes orientation over time. Data obtained from a single cross for cal1 mutant and for wild type (wt), with a use of different fly line expressing Mis12::GFP. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size, filmed 1 time point/min. Z sections were cropped individually for every time point shown. Contrast was adjusted for full projection of individual image. Scale 5 μm.

A Cen3-Cen3 distance [μm] Cen3 merge wt cal1 DNA wt (n=10) N=1 cal1 (n=46) N=2 B % oocytes % oocytes 100 100 80 80 60 60 40 40 20 20 wt (n=10) N=1 cal1 (n=46)N=2 wt (n=10)N=1 cal1 (n=37)N=3 uncondensed-looking chromosomes congressed chromosomes uncongressed chromosomes Fig.32. cal1 mutation decreases the frequency of homologous centromeres biorientation and chromosome congression. FRT cal1 flies were crossed with FRT ovoD flies and mutant oocytes were analysed 5 days after heat shock of eclosed progeny. A. Oocytes subjected to fluorescence in situ hybridization (FISH) to mark the centromere 3 and stained with DAPI. Image of chromosomes with minimal Cen3-Cen3 distance. Black bars in the chart represent Cen3-Cen3 distance [μm] of homologs. Red bar is a median. cal1 mutation results in significantly decreased Cen3-Cen3 distance (p<0.01). B. Chromosome arrangement was assessed by DAPI staining in oocytes subjected to FISH (left) or in immunostained oocytes (right) and categorized as indicated. cal1 mutation, results in increased frequency of uncongressed chromosomes. Uncondensed-looking chromosomes defined by obvious lack of chromatin compaction. N value represents number of fly crosses made to obtain the data presented. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 5 μm.

α-tubulin dTACC DNA merge wt cal1 % oocytes 100 80 60 single classic-shape spindle 40 single mal-shaped spindle multiple spindles 20 wt (n=10) cal1 (n=33) Fig.33. cal1 mutation leads to formation of multiple spindles. FRT cal1 flies were crossed with FRT ovoD flies and mutant oocytes were fixed 5 days after heat shock of eclosed progeny. Mutant oocytes were immunostained with Ab agains α-tubulin and dTACC. Chromosomes were stained with DAPI. In contrast to in wild type, cal1 mutant leads to appearance of multiple spindles in 20% of oocytes analysed. Data obtained from 3 crosses to generate homozygous cal1 mutant and from a single batch of wt. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

nanos GAL4 mat α-tubilin GAL4 (V37) mat α-tubilin GAL4 (V2H) 25°C 18°C 25°C 18°C 25°C eggs ++ eggs ++ eggs ++ eggs ++ eggs ++ wt larvae ++ larvae ++ larvae ++ larvae ++ larvae ++ cenp-C RNAi eggs 0 eggs ++ eggs ++ ND ND larvae 0 larvae 0 larvae 0 mis12 RNAi eggs ++ eggs + eggs ++ larvae + ND larvae ++ larvae ++ ND nuf2 RNAi eggs ++ eggs + eggs + eggs ++ ND larvae 0 larvae 0 larvae + larvae ++ spc105 RNAi eggs 0 eggs ++ eggs ++ eggs ++ ND larvae 0 larvae 0 larvae 0 larvae + ndc80 RNAi eggs ++ eggs ++ eggs ++ ND ND larvae + larvae 0 larvae + cenp-meta RNAi eggs ++ eggs ++ eggs ++ eggs ++ eggs ++ larvae 0 larvae 0 larvae 0 larvae 0 larvae + Fig.34. Germline-specific RNAi of kinetochore components results in female sterility or reduced fertility. Around 15 RNAi flies of each kinetochore component derived from a cross of UASp-RNAi flies with flies carrying a nanos and maternal α-tubulin driver (V37) or (V2H) were maturated at 25°C or 18°C for 8 and over 8 days, respectively. Effect on eggs laying and fly development was assessed in the heterozygotes. Lack of development suggests a candidate with defective female meiosis. Production of eggs gives a chance for phenotype analysis. nanos and (V37) drivers resulted in significantly reduced fertility in most RNAi flies. However, RNAi driven by (V37), but not by nanos, allowed for production of eggs in all RNAi flies. Data obtained from two crosses to generate RNAi flies. ND-not determined.

kinetochore foci number
A Nsl1R::GFP H2Av::mRFP merge Ratio of kinetochore signal intensity to the background signal intensity wt 1.8 1.6 luciferase RNAi 1.4 1.2 1 spc RNAi 0.8 wt (n=28) luciferase RNAi (n=28) spc105 RNAi (n=27) B % oocytes with kinetochore foci after NEB 100 80 60 40 20 20% LP (n=26) 50% LP (n=14) 20% LP (n=28) 50% LP (n=15) 50% LP (n=9) 100% LP (n=9) wt luciferase RNAi spc105 RNAi kinetochore foci number >8 8 7 6 5 4 3 2 1 Fig.35. Kinetochore is not fully assembled in spc105 RNAi. Wild type, UASp-spc105 and control RNAi (luciferase RNAi) flies were crossed with a recombinant of maternal α-tubulin (V37) with UASp Nsl1R::GFP and H2Av::mRFP (expressed under their own promoter) markers and the heterozygous progeny was maturated 3-4 days at 25°C. A Intensity ratio of Nsl1R::GFP signal at the kinetochore to the background intensity was measured in oocytes of all stages after NEB (assessed by H2Av::mRFP absence from the nucleus area). 4 kinetochore foci (in one case just 3) from each oocyte were included in the measurement. spc105 RNAi results in a significantly reduced Nsl1R::GFP signal intensity comparing to in wild type and luciferase RNAi controls (p<0.01). Images for the measurement were obtained at 250 ms exposure time and 50% 488 laser power (LP). The error bars represent the standard error of the mean. B The number of Nsl1R::GFP foci at the chromosome area in oocytes was counted for different 488 LP used to obtain the images. spc105 RNAi does not decrease the number of visible Nsl1R::GFP foci comparing to in controls. Data obtained from two crosses to induce Nls1R and luciferase RNAi and a single cross to generate wild type flies. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 5 μm.

A GFP-Rod H2Av::mRFP merge wt spc105 RNAi B % oocytes with GFP-Rod signal at kinetochores and microtubules 100 80 60 40 20 kt mt kt mt kt mt kt mt 20% LP 20% LP 50% LP 100% LP n=12 n=14 n=39 n=15 wt spc105 RNAi Fig.36. GFP-Rod does not stream along microtubules in spc105 RNAi oocytes. Wild type (wt) and UASp-spc105 RNAi flies were crossed with a recombinant of maternal α-tubulin (V37) with GFP-Rod and H2Av::mRFP (both expressed under its own promoters) markers and the heterozygous progeny was maturated 3-4 days at 25°C. A In contrast to in wt, GFP-Rod signal is invisible in spc105 RNAi oocytes exposed to 20% laser power (LP). B GFP-Rod presence at microtubules and kinetochores in oocytes was assessed for analysed on images obtained using different 488 LP. Exposure time 250 ms. In contrast to in wild type, in spc105 RNAi, GFP-Rod is not frequently visible on microtubules, independently of 488 LP used. In spc105 RNAi, GFP-Rod foci can be sometimes be observed when stronger 488 LP is used. Analysis included oocytes of all stages after nuclear envelope breakdown. Oocytes of stage 14 in wt accounted for 36% of oocytes exposed to 20% LP. For spc105 RNAi stage 14 contributed in 50%, 51% and 33% to the total number of oocytes exposed to 20%, 50% and 100% LP, respectively. Data obtained from 2 crosses to induce spc105 RNAi and a single cross to generate control flies. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

centromere foci number
A After NEB Before NEB GFP-CID H2Av::mRFP merge GFP-CID merge wt wt (n=2) luciferase RNAi spc105 RNAi (n=6) spc RNAi centromere foci after NEB % oocytes 100 80 centromere foci number 5 60 >8 4 8 3 40 7 2 20 6 1 wt (n=12) luciferase RNAi (n=12) spc105 RNAi (n=15) B centromere orientation after NEB % oocytes 100 centromeres positioned exterior of chromosomes, with equal number of foci either side (3+4 or 4+4) 80 60 centromeres positioned exterior of chromosomes, with incorrect number of foci on either side 40 20 centromeres within the chromatin mass wt (n=12) luciferase RNAi (n=14) spc105 RNAi (n=15) Fig.37. In spc105 RNAi oocytes, centromeres are de-clustered after nuclear envelope breakdown but not positioned at the exterior of chromatin mass. Figure description on the following page.

Fig.37. In spc105 RNAi oocytes, centromeres are de-clustered after nuclear envelope breakdown but not positioned at the exterior of chromatin mass. Wild type, UASp-spc105 and control RNAi (Luciferase RNAi) flies were crossed with a recombinant of maternal α-tubulin (V37) GFP-CID H2Av::mRFP (both expressed under their own promoters) and the heterozygous progeny was maturated 3-4 days at 25°C. A As in the wild type, GFP-CID appears most often as 8 foci at chromosomes after nuclear envelope breakdown (NEB), while centromeres are clustered before NEB. B Unlike in wild type, GFP-CID foci are positioned within chromatin mass. Data obtained from two crosses to induce spc105 RNAi and a single cross to generate control flies. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 5 μm. % oocytes with congressed chromosomes 100 80 60 40 20 (n=12) N=1 (n=13) N=1 (n=25) N=1 (n=30) N=1 (n=29) N=2 (n=15) N=2 (n=57) N=2 (n=16) N=2 GFP-CID GFP-Rod Nsl1R::GFP GFP-CID Nsl1R::GFP GFP-CID GFP-Rod Nsl1R::GFP wt luciferase RNAi spc105 RNAi Fig.38. In spc105 RNAi chromosomes are congressed. Wild type, UASp-spc105 and control RNAi (luciferase RNAi) flies were crossed with a recombinant of maternal α-tubulin (V37) with indicated markers and the heterozygous progeny was maturated 3-4 days at 25°C. Chromosome congression was assessed based on wt and spc105 RNAi and control RNAi oocytes co-expressing either GFP-CID, GFP-Rod (both expressed under their own promoters) or Nsl1R::GFP and H2Av::mFRP (expressed under its own promoter). As in controls, in spc105 RNAi, the chromosomes are congressed. Analysis included oocytes of all stages after NEB. Oocytes of stage 14 accounted for around 50% of spc105 RNAi, luciferase RNAi and wt oocytes. N value represents number of fly crosses made to obtain the data presented. n value represents number of oocytes analysed.

CHROMATIN SHAPES classic ellipse sphere % oocytes with different chromatin shapes 100 80 60 40 20 (n=12) N=1 (n=13) N=1 (n=25) N=1 (n=30) N=1 (n=29) N=2 (n=15) N=2 (n=57) N=2 (n=16) N=2 GFP-CID GFP-Rod Nsl1R::GFP GFP-CID Nsl1R::GFP GFP-CID GFP-Rod Nsl1R::GFP wt luciferase RNAi spc105 RNAi classic ellipse or sphere others Frequency of spherical and ellipsoid chromatin shapes in different stages of Spc105 RNAi oocytes % oocytes 100 80 60 40 20 GFP-CID (n=5) GFP-Rod (n=10) Nsl1R::GFP (n=7) GFP-CID (n=3) GFP-Rod (n=20) Nsl1R::GFP (n=4) GFP-CID (n=7) GFP-Rod (n=27) Nsl1R::GFP (n=5) STAGE: early 13 late 13 14 classic ellipse sphere others Fig.39. In Spc105 RNAi chromosomes do not adopt the classic shape but become compacted into a spherical shape in later oocyte stages. Figure description on the following page

Fig.39. In spc105 RNAi chromosomes do not adopt the classic shape but become compacted into a spherical shape in later oocyte stages. Wild type, UASp-spc105 and control RNAi (luciferase RNAi) flies were crossed with a recombinant of maternal α-tubulin (V37) with indicated markers and the heterozygous progeny was maturated 3-4 days at 25°C. Chromosome shape was assessed based on wt and spc105 RNAi and control RNAi flies co-expressing either GFP-CID, GFP-Rod (both expressed under their own promoters) or Nsl1R::GFP together with H2Av::mFRP (expressed under its own promoter). The classification of chromosome shape is based only on H2Av::mRFP signal analysis. Typical shapes (classic) for wild type and control RNAi oocytes are rarely found in spc105 RNAi oocytes. Instead, chromatin shape in spc105 RNAi oocytes frequently resembles a sphere or an ellipse (upper chart). The ellipsoid shape is more common for younger oocyte stages, while sphere shape predominates in later oocyte stages (lower chart). N value represents number of fly crosses made to obtain the data presented. n value represents number of oocytes analysed.

Cen3-Cen3 distance [μm] V37/ Gal4 RNAi (n=27) V37/ Mis12 RNAi (n=65) V37/ Ndc80 RNAi (n=67) V37/ Spc105 RNAi (n=42) V2H/ Spc105 RNAi (n=35) Fig.40. Centromeres of homologs are variably positioned within the chromatin mass after knockdown of kinetochore components. Figure description on the following page.

Fig.40. Centromeres of homologs are variably positioned within the chromatin mass after knockdown of kinetochore components. Heterozygotes of UASp-RNAi and maternal α-tubulin drivers (V37) or (V2H) were maturated 4-5 days at 25°C. Oocytes were fixed and subjected to fluorescence in situ hybridization to mark the centromere 3 (Cen3). Chromosomes were stained with DAPI. Black bars represent Cen3-Cen3 distance [μm] of homologs. Red bar is a median. When more than 2 Cen3 foci were visible, the distance measured was between the middle of the closest 2 foci and the more distant foci. In wild type (wt) oocytes, almost all Cen3-Cen3 values are above 2 μm. Low Cen3-Cen3 distance values appear in RNAis of kinetochore proteins. spc105 RNAi under (V37) driver significantly reduced the Cen3-Cen3 distance values, comparing to wt (p<0.01). This effect of spc105 RNAi effect was alleviated using (V2H) driver, but still significant (p<0.01). The Cen3-Cen3 distance values in spc105 RNAi are evenly distributed, suggesting that the Cen3-Cen3 distance is variable. However, the distribution of Cen3-Cen3 distance is constrained to values between 0 and 3 μm, unlike in the wt. Data obtained from a single cross to induce each RNAi. n value represents number of oocytes analysed.

[μm] spindle length chromosome spread spindle length chromosome spread wt (n=60) spc105 RNAi (n=23) Fig.41. Dysfunction of kinetochores leads to hypercongressed chromosomes. Heterozygotes of maternal α-tubulin driver (V37) and UASp-spc105 RNAi were maturated 4-5 days at 25°C. Oocytes were fixed and immunostained with Ab agains α-tubulin. Chromosomes were stained with DAPI. Black bars represent the measured distances [μm]. Red bar is a median. spc105 RNAi results in a significant decrease in chromosome span (p<0.01), while the range of spindle length remains similar to in the wild type.(p>0.01). Data obtained from two crossses to generate control oocytes (V37/+) and a single cross to induce Spc105 RNAi. n value represents number of oocytes analysed.

α-tubulin DNA CID merge wt spc105 RNAi % oocytes 100 80 60 classic-shape spindle 40 unfocused spindle 20 wt (n=60) spc105 RNAi (n=23) Fig.42. Dysfunction of kinetochores leads to unfocused spindles. Heterozygotes of maternal α-tubulin driver (V37) and UASp-spc105 RNAi were maturated 4-5 days at 25°C. Oocytes were fixed and immunostained with Ab agains α-tubulin and CID. Chromosomes were stained with DAPI. spc105 RNAi leads to increased frequency of unfocused spindles. Spindle length measurement for spindles with unfocused poles included longest microtubule bundle in the vicinity of chromosomes. Data obtained from two crosses to generate control oocytes (V37/+) and a single cross to induce spc105 RNAi. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

GFP-Rod RCC1::mCherry merge wt cenp-meta RNAi Frequency of GFP-Rod accumulation at kinetochores % oocytes 100 80 60 40 20 wt (n=40) cenp-meta RNAi (n=19) wt (n=8) cenp-meta RNAi (n=12) STAGE 13 14 Fig.43. Depletion of Cenp-meta disables formation of robust kinetochore-microtubule attachments. Heterozygotes of cenp-meta RNAi nanos GAL4 RCC1::mCherry GFP-Rod (GFP-Rod expression under its own promoter) and wild type flies were maturated 4-6 days at 18°C. cenp-meta RNAi results in increased frequency of oocytes with GFP-Rod accumulation at kinetochores in oocytes of stage 13 and 14, as compared to wild type. The frequency of accumulation decreases in later oocyte stages, suggesting that some kinetochores become eventually attached, but not all. Data based on three crosses to generate control flies and two crosses to induce cenp-meta RNAi. cenp-meta RNAi and control oocytes were analysed at different times. Z sections for the images of oocytes stage 14 were taken at 0.5 μm step size. Scale 10 μm.

RCC1::mCherry GFP::α-Tubulin merge wt cenp-meta RNAi 1 2 % oocytes 100 80 60 40 20 wt (n=27) cenp-meta RNAi (n=9) wt (n=23) cenp-meta RNAi (n=26) STAGE 13 14 congressed chromosomes uncongressed chromosomes within one spindle uncongressed chromosomes in multiple spindles Fig.44. Inability to establish robust kinetochore-microtubule attachments results in uncongressed chromosomes. Heterozygotes of cenp-meta RNAi nanos GAL4 RCC1::mCherry GFP::α-tubulin and wild type flies were maturated 4-6 days at 18°C. cenp-meta RNAi results in increased frequency of oocytes with uncongressed chromosomes within one spindle (image 1), but particularly within multiple spindles (image 2), as compared to wild type. This frequency is similar between stages 13 and 14 of oocytes. Data based on two crosses to generate control flies and two crosses to induce cenp-meta RNAi. cenp-meta RNAi and control oocytes were analysed at different times. n value represents number of oocytes analysed. Z sections for the images of oocytes stage 14 were taken at 0.5 μm step size. Scale 10 μm.

% stage 14 oocytes with correct orientation
Mis12::GFP RCC1::mCherry merge wt c-met c-met RNAi % stage 14 oocytes with correct orientation 100 80 60 40 20 wt (n=32) cenp-meta RNAi (n=10) c-met (n=4) N=2 N=1 N=1 Fig.45. Inability to establish robust kinetochore-microtubule attachments results in maloriented chromosomes. c-met FRT flies were crossed with FRT ovoD flies and homozygous mutant c-met FRT/c-met FRT; nanos GAL4 UASp RCC1::mCherry Mis12::GFP/+ oocytes were analysed 3 days after maturating females at 18°C. Heterozygote cenp-meta RNAi nanos GAL4 RCC1::mCherry Mis12::GFP oocytes were maturated 4-6 days at 18°C before oocyte analysis. Induced site-directed mutagenesis and cenp-meta RNAi result in decreased frequency of stage 14 oocytes with correctly oriented chromosomes. Mutants, cenp-meta RNAi and control oocytes were analysed at different times. Orientation of chromosomes was assessed based on pictures and onsets of the films. N value represents number of fly crosses made to obtain the data presented. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

% stage 14 oocytes with stable orientation
% stage 13 oocytes with kinetochores changing position within chromatin mass while GFP-Rod is accumulated at kinetochores B % stage 14 oocytes with stable orientation 100 100 80 80 60 60 40 40 20 20 wt (n=24) t=790 min N=2 cenp-meta RNAi (n=6) t=252 min N=1 wt (n=13) t=620 min N=3 cenp-meta RNAi (n=7) t=379 min N=2 C wt GFP-Rod 18 min 29 min 30 min 34 min 48 min merge RCC1::mCherry cenp-meta RNAi GFP-Rod 6 min 11 min 21 min 26 min 34 min merge GFP-Rod 40 min 48 min 54 min 64 min 106 min merge RCC1::mCherry Fig.46. Inability to establish robust kinetochore-microtubule attachments abolishes chromosome re-orientation. Figure description on the following page.

Fig.46. Inability to establish robust kinetochore-microtubule attachments abolishes chromosome re-orientation. Heterozygotes of nanos GAL4 RCC1::mCherry Mis12::GFP (A) and nanos GAL4 RCC1::mCherry GFP-Rod (B and C; GFP-Rod expressed under its own promoter) were maturated 4-6 days at 18°C. A. Stage 14 oocytes of nanos GAL4 RCC1::mCherry Mis12::GFP heterozygotes have chromosomes with stable orientation. cenp-meta RNAi and control oocytes were analysed at different times. N value represents number of fly crosses made to obtain the data presented. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size, filmed 1 time point/min for at least 20 min. B Chromosomes of stage 13 oocytes, with GFP-Rod accumulated at kinetochores rarely change orientation, as opposed to wild type oocytes, in which GFP-Rod accumulation seems coupled to chromosome change of orientation. Analysis did not include oocytes in which chromosomes looked de-condensed. cenp-meta RNAi and control oocytes were analysed at different times. Z sections for the images were taken at 0.5 μm step size, filmed 2 time point/min for at least 20 min. C After the initiation of an abrupt chromosome movement, accompanying nuclear envelope breakdown (NEB; t0), most kinetochores in cenp-meta RNAi oocytes accumulate GFP-Rod and do not change orientation, while chromosomes separate. To the contrary, frequent kinetochore position change within chromatin mass is observed in wild type oocytes after NEB. Data based on two oocytes from two independent crosses to generate cenp-meta RNAi flies and on 13 oocytes from three independent crosses to generate control flies. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min at least 40 min. Z sections were cropped individually for every time point shown. Scale 10 μm.

weak and unstable kinetochore-microtubule attachment
WILD TYPE OOCYTE STAGE 13 OOCYTE STAGE 14 DE-CONGRESSION RE-CONGRESSION weak and unstable kinetochore-microtubule attachment stable kinetochore-microtubule attachment DEPLETION OF CENP-META DE-CONGRESSION destabilized kinetochore-microtubule attachment destabilized kinetochore-microtubule attachment DEPLETION OF KINETOCHORE STRUCTURAL PROTEINS (Spc105) HYPER-CONGRESSION no kinetochore-microtubule attachment no kinetochore-microtubule attachment chromosomes spindle kinetochores unstable attachment and direction of kinetochore pulling nuclear envelope stable attachment and direction of kinetochore pulling Fig.47. Model for the effect of kinetochore dysfunction on chromosome positioning within the spindle in Drosophila oocytes. Figure description on the following page.

Fig.47. A model for the effect of kinetochore dysfunction on chromosome positioning within the spindle in Drosophila oocytes. In wild type, weak and unstable kinetochore-microtubule attachments lead separation of chromosomes from the chromosome mass and change of orientation. Bi-orientation and congression are followed by stabilization of kinetochore-microtubule attachments. If kinetochore-microtubule interactions are additionally destabilized (Cenp-meta depletion), kinetochores lead de-congression but are unable to lead orientation change and re-congression. Complete dysfunction of kinetochores (depletion of kinetochore structural proteins) disables kinetochore-microtubule attachments and so chromosomes do not de-congress and change orientation after de-clustering. Polar ejection force, imbalanced by kinetochore pulling, leads to chromosome hyper-congression.

% oocytes with wide chromosome span in different oocyte stages
RCC1::mCherry GFP::α-tubulin merge wt wac % oocytes with wide chromosome span in different oocyte stages 100 80 60 40 20 wt wac wt wac wt wac (n=14) (n=27) (n=27) (n=28) (n=62) (n=82) STAGE early 13 late 13 14 8-10 μm >10 μm Fig.48. Augmin is crucial for limiting chromosome span particularly in young spindles. wac RCC1::mCherry GFP::α-tubulin nanos GAL4/wac and RCC1::mCherry GFP::α-tubulin nanos GAL4 heterozygotes were maturated 5 days at around 21°C. Chromosome span in oocytes was measured on the spindle axis. wac mutation results in increased proportion of oocytes with chromosomes spanning over 10 μm. Extensive chromosome span is found particularly frequently in younger oocyte stages, indicating that Augmin function is particularly important in that time. Data based on three crosses to generate the wac mutant and control flies. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

FRAP No FRAP -5 sec 0 sec 10 sec 20 sec 30 sec 40 sec 50 sec 60 sec 70 sec 80 sec Wac::GFP Dynamics of Wac::GFP signal recovery after photobleaching in syncytial mitoses mitosis (this study) meiosis (Colombie) [sec] n-=11-17 n-=2-8 Fig.49. Wac::GFP is less stable on the mitotic spindle than on the spindle in oocytes. Heterozygotes of RCC1::mCherry Wac::GFP nanos Gal4 were maturated 3-5 days at 25°C. Embryos laid within 2 hrs were used for the experiment. Single spindle at prophase/ metaphase was exposed to 100% laser power (red rectangle) and imaged until anaphase onset. Fluorescence recovery after photobleaching (FRAP) of Wac::GFP signal on syncytial spindles was measured (blue dots and blue line of the total fit) and compared to FRAP result in oocytes, obtained by Colombie (red dashed line, schamatic). Corrected and normalized values for Wac::GFP dynamics in mitosis were plotted as a function of time [sec] as dots. Total fit is the blue line. Over 60% of Wac::GFP signal recovers from photobleaching within the measured time (t1/2= 20 sec). This is in contrast to only around 20% Wac::GFP signal recovered within that time in meiosis. The difference in Wac::GFP turnover suggests that in meiosis Wac::GFP is more stably bound to spindle microtubules than in mitosis. Data based on three independent crosses to generate the heterozygotes. Z sections for the images were taken at 1 μm step size, at 5 sec interval. n value represents number of oocytes analysed. Scale 10 μm.

MITOSIS No FRAP FRAP -5 sec 0 sec 10 sec 20 sec 30 sec 40 sec 50 sec 60 sec 70 sec 80 sec GFP::α-Tubulin MEIOSIS No FRAP -5 sec 15 sec 35 sec 55 sec 75 sec 95 sec FRAP -5 sec 0 sec 10 sec 20 sec 30 sec 40 sec 50 sec 60 sec 70 sec 80 sec 90 sec 100 sec Dynamics of GFP::α-Tubulin signal recovery after photobleaching Meiosis Syncytial mitosis [sec] [sec] n=20 n=14-16 n=2-10 Fig.50. GFP::α-tubulin turnover is comparable between mitotic and meiotic spindles. Fluorescence recovery after photobleaching (FRAP) experiment was done on embryos and oocytes derived from heterozygotes of RCC1::mCherry GFP::α-tubulin nanos GAL4. Flies for FRAP in embryos were maturated 3-5 days at 25°C and flies for FRAP in oocytes were maturated 5 days at 25°C. GFP::α-tubulin signal intensity in a region exposed to 100% laser power (red rectangle), was corrected against non-bleached spindles (neighbouring non-bleached spindle for embryos and non-bleached spindle in an oocyte). Normalized values were plotted as a function of time [sec] as dots. Total fit is the blue line. Bleached GFP::α-tubulin signal in mitosis and meiosis recovers to a comparable extent in meiosis and mitosis, with t1/2=35 sec for meiosis and t1/2=17 sec for mitosis. Data based on three independent crosses to generate the heterozygotes. Z sections for the images were taken at 1 μm step size, at 5 sec interval. n value represents number of oocytes analysed. Scale 10 μm.

transient association of Augmin with microtubules
MITOSIS transient association of Augmin with microtubules amplification of microtubules FEMALE MEIOSIS stable association of Augmin with microtubules generation of microtubules from the poles and chromosome congression centrosomes direction of microtubule polymerization Augmin microtubules chromosomes Fig.51. A model of Augmin function in Drosophila oocytes. In mitosis, Augmin transiently binds to the spindle microtubules throughout the spindle and amplifies microtubules. In acentrosomal spindle, Augmin binds stably to the polar regions on the spindle and generates new microtubules. Stable association of Augmin to the spindle generates polar ejection force, which limits chromosome span within the spindle.

A % oocytes with maloriented chromosomes 100 Mis12::GFP RCC1::mCh merge wt 80 60 40 sentin 20 wt (n=26) sentin (n=11) wt (n=31) sentin (n=29) stage 13 stage 14 B % oocytes with stable orientation of maloriented chromosomes within 20 min % oocytes with stable chromosome orientation 100 100 80 80 60 60 40 40 20 20 wt (n=25) t=855 min sentin (n=11) t=424 min wt (n=25) t=697 min sentin (n=19) t=867 min wt (n=5) t= 100 min sentin (n=10) t=200 min sentin (n=12) t=240 min stage 13 stage 14 stage 13 stage 14 Fig.52. The sentin mutation results in stably maloriented chromosomes. Wild type (wt) Mis12::GFP nanos GAL4 RCC1::mCherry heterozygotes and sentin/Df mutant flies, expressing the same markers, were maturated 4-7 days at 18°C. A Oocytes with correct number of kinetochores on either side of chromatin mass (4+4 or 3+4) were counted. sentin mutation results in high frequency of maloriented chromosomes. Data based on images and first time point of films. B Position of kinetochores within chromatin mass was assessed for oocytes of stage 13 and 14 (excluding oocytes found before nuclear envelope breakdown) for minimum time of 20 min for each oocyte, 1 time point/minute. Analysis did not include oocytes with scattered chromosomes. In the sentin mutant, position of kinetochores on chromosome sides is stable. Orientation of maloriented chromosomes in sentin mutant is more stable than in wt early oocyte stages. Data obtained from two crosses to generate wt and sentin mutant oocytes. The sentin mutant and wild type oocytes were analysed at different times. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 5 μm.

oocyte 1 oocyte 2 oocyte 3 oocyte 4 oocyte 5 oocyte 6 oocyte 7 oocyte 8 chromosome span location of kinetochores 2 min apart Fig.53. In wild type, kinetochores frequently change location within chromatin span after nuclear envelope breakdown (NEB). Mis12::GFP nanos GAL4 RCC1::mCherry heterozygotes were maturated 4-7 days at 18°C. Kinetochore behaviour in oocytes was analysed after full accumulation of Mis12::GFP signal at kinetochores. Rough location of kinetochores (black) on the chromosome mass (red) assessed every 2 min is indicated. Distance of kinetochore location from the main chromatin mass represents small fourth chromosome or a thin chromatin protrusion. Kinetochores frequently change position within the span of chromatin mass after NEB. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min.

oocyte 1 oocyte 2 oocyte 3 oocyte 4 oocyte 5 oocyte 6 chromosome span location of kinetochores 2 min apart Fig.54. In the sentin mutant, kinetochores location on chromatin mass is determined quickly after nuclear envelope breakdown (NEB). Figure desciption on the following page.

Fig.54. In the sentin mutant, kinetochores location on chromatin mass is determined quickly after nuclear envelope breakdown (NEB). sentin Mis12::GFP nanos GAL4 RCC1::mCherry/sentin flies were maturated 4-7 days at 18°C. Kinetochore behaviour in oocytes was analysed after full accumulation of Mis12::GFP signal at kinetochores. Rough location of kinetochores (black) on the chromosome mass (red) assessed every 2 min is indicated. Distance of kinetochore location from the main chromatin mass represents small fourth chromosome or a thin chromatin protrusion tipped with kinetochore. Kinetochores in the sentin mutant rarely change position within the span of chromatin mass after NEB. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min.

wt 0 min 19 min 23 min 26 min 52 min 96 min 122 min RCC1::mCh Mis12::GFP merge sentin 0 min 10 min 16 min 26 min 31 min 50 min 97 min RCC1::mCh Mis12::GFP merge % oocytes in which chromosomes loose association after NEB 100 80 60 40 20 wt n=8 sentinn=6 Fig.55. Chromosomes do not loose association in the sentin mutant during spindle assembly. Mis12::GFP nanos GAL4 RCC1::mCherry heterozygotes and sentin Mis12::GFP nanos GAL4 RCC1::mCherry/sentin flies were maturated 4-7 days at 18°C. Oocytes were counted based on chromosome behaviour after nuclear envelope breakdown (NEB). While in wild type oocytes chromosome often lose association during chromosome positioning, in the sentin mutant no individual chromosomes are visible. Z sections for the images were taken at 0.8 μm step size, filmed 1 time point/min. Scale 5 μm.

A GFP-Rod merge GFP-Rod merge wt sentin Frequency of GFP-Rod accumulation at kinetochores % oocytes 100 80 60 40 20 wt (n=25) sentin (n=20) wt (n=10) sentin (n=12) wt (n=18) sentin (n=21) STAGE early 13 late 13 14 B Frequency of kinetochore-microtubule detachment % oocytes 100 80 60 40 20 wt (n=18) t=756 min sentin (n=16) t=560 min wt (n=13) t=454 min sentin (n=15) t=480min stage 13 stage 14 Fig.56. In the sentin mutant, kinetochores are stably attached by microtubules. Figure description on the following page.

Fig.56. In the sentin mutant, kinetochores are stably attached by microtubules. Wild type (wt) Mis12::GFP nanos GAL4 RCC1::mCherry heterozygotes and sentin/Df mutant flies, expressing the same markers, were maturated 4-7 days at 18°C. A Oocytes with GFP-Rod accumulation at kinetochores were counted in oocytes of different stages after nuclear envelope breakdown (NEB; excluding oocytes found before NEB). The sentin mutation results in low frequency of oocytes with GFP-Rod accumulation at kinetochores, suggesting fast establishment of stable kinetochore-microtubule attachments, even faster than in wt oocytes. Data based on images and first time point of films. Images were taken at 0.5 μm step size. Scale 10 μm. B GFP-Rod accumulation events were analysed in oocytes of stage 13 and 14 (excluding oocytes found before NEB), with all kinetochores attached at the onset of the film. Minimum time of the film 20 min for each oocyte, filmed 1 time point/minute at 0.5 μm step size. Similarly to in wt, once kinetochores are attached in the sentin mutant, the attachment persists. Data obtained from three crosses to generate wt and sentin mutant oocytes with fluorescent markers. sentin mutant and wild type oocytes were analysed at different times. n value represents number of oocytes analysed.

% stage 14 oocytes with congressed chromosomes wt 100
Mis12::GFP RCC1::mCh merge B % stage 14 oocytes with congressed chromosomes wt 100 80 sentin 60 40 incenp 20 wt (n=31) sentin (n=29) incenp (n=46) C % stage 14 oocytes with correct chromosome orientation D % stage 14 oocytes with kinetochore cluster on chromosome side 100 100 80 80 60 60 40 40 20 20 wt (n=31) sentin (n=29) incenp (n=46) wt (n=31) sentin (n=29) incenp (n=46) E % oocytes 100 80 60 40 20 wt (n=26) sentin (n=11) incenp (n=9) wt (n=31) sentin (n=29) incenp (n42) stage 13 stage 14 spherical shape of the central chromatin mass without protrusions spherical shape of the central chromatin mass with protrusions Fig.57. Malorientation of chromosomes, common for the sentin and incenp mutants is associated with different geometrical arrangement of kinetochores and chromatin in these two mutants. Figure description on the following page.

% stage 14 oocytes with robust GFP-Rod localization on spindle
Fig.57. Malorientation of chromosomes, common for the sentin and incenp mutants is associated with different geometrical arrangement of kinetochores and chromatin in these two mutants. Wild type (wt) Mis12::GFP nanos GAL4 RCC1::mCherry heterozygotes, as well as sentin/Df and incenp/incenp mutant flies, expressing the same markers, were maturated 4-7 days at 18°C. Oocytes were counted based on chromosome position (B) and shape (E) and kinetochore position on chromatin mass (C, D). A Live oocytes of stage 14. Z sections for the images were taken at 0.5 μm step size. Scale 5 μm. B incenp mutant oocytes have chromosomes less frequently congressed than wt and sentin mutant oocytes. C incenp mutant oocytes have maloriented chromosomes. D In the sentin mutant, kinetochores are frequently clustered on at least one side of chromosomes, rather than evenly spaced on chromatin side, as in incenp mutant. E Shape of the chromatin mass in the sentin mutant is often spherical. Protrusions from the chromatin sphere are often seen, particularly in stage 13. This effect is very rarely observed in the incenp mutant. Data based on images and first time point of films. Data obtained from two crosses to generate wt as well as sentin and incenp mutant oocytes. The mutants and wild type oocytes were analysed at different times. n value represents number of oocytes analysed. GFP-Rod RCC1:mCherry merge wt % stage 14 oocytes with robust GFP-Rod localization on spindle 100 80 sentin 60 40 incenp 20 wt (n=18) sentin (n=21) incenp (n=30) Fig.58. The sentin and incenp mutants have a different effect on distribution of GFP-Rod signal. Wild type (wt) GFP-Rod nanos GAL4 RCC1::mCherry heterozygotes, as well as sentin/Df and incenp/incenp mutant flies, expressing the same markers, were maturated 4-7 days at 18°C. Oocytes were counted based on GFP-Rod abundance on division apparatus. In wt and sentin mutant oocytes of stage 14, GFP-Rod signal is similarly robustly localized on microtubules. To the contrary, in incenp mutant oocytes, GFP-Rod signal abundance is very reduced. Data based on images and first time point of films. Data obtained from three crosses to generate wt, sentin mutant or incenp mutant oocytes with fluorescent markers. The mutants and wild type flies were analysed at different times. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

Sentin::GFP RCC1::mCherry merge Stage early 13 Stage 14 % oocytes Sentin localization 100 80 60 throughout the spindle 40 at spindle poles 20 early 13 (n=13) late 13 (n=5) 14 (n=12) Fig.59. Sentin::GFP concentrates on the spindle poles over time. Heterozygote females of Sentin::GFP and RCC1::mCherry nanos GAL4 were maturated for 5 days at 18°C. Oocytes were counted based on Sentin::GFP localization pattern in different oocyte stages after nuclear envelope breakdown. Sentin localizes throughout the spindle in stage 13 and to the spindle poles in stage 14. Data obtained from a single cross. n value represents number of oocytes analysed.

[μm] spindle length chromosome span spindle length chromosome span spindle length chromosome span spindle length chromosome span wt (n=7) wac (n=23) sentin (n=34) sentin wac (n=40) Fig.60. The sentin wac double mutant is more similar to the single sentin mutant than to the single wac mutant in terms of spindle length and chromosome span. Figure description on the following page.

Fig.60. The sentin wac double mutant is more similar to the single sentin mutant than to the single wac mutant in terms of spindle length and chromosome span. Wild type (wt) females, as well as sentin wac/sentin, wac sentin/wac sentin and wac/wac mutant females were maturated 4 days at 25°C. Oocytes were fixed and immunostained to visualize α-tubulin. Chromosomes were stained with DAPI. Black bars represent the measured distances [μm] of spindle length and chromosome span. Red bar is a median. The sentin wac double mutant has spindle length values and chromosome span values similar to in the sentin single mutant (p>0.01) and significantly different from in the wac single mutant (p>0.01). Data obtained from three independent batches of sentin wac double mutant and one batch of wac and sentin single mutants, as well as of wt. n value represents number of oocytes analysed.

α-tubulin dTACC DNA merge wt wac sentin sentin wac Frequency of split poles % oocytes 100 80 60 40 20 wt (n=7) wac (n=23) sentin (n=34) sentin wac (n=40) Fig.61. The sentin wac double mutant produces more split poles than the sentin and wac single mutants. Figure description on the following page

Fig.61. sentin wac double mutant produces more split poles than the sentin and wac single mutants. Wild type (wt) females, as well as sentin wac/sentin, wac sentin/wac sentin and wac/wac mutant females were maturated 4 days at 25°C. Oocytes were fixed and immunostained to visualize α-tubulin and d-TACC. Chromosomes were stained with DAPI. . The percentage of oocytes with split poles was counted based on α-tubulin staining only. Sentin wac double mutant results in increased frequency of split poles than sentin and wac single mutants, indicating an synergistic effect of these two mutants. Data obtained from three batches of sentin wac double mutant and one batch of wac and sentin mutants, as well as for wt. n value represents number of oocytes analysed. Z sections for the images were taken at 0.5 μm step size. Scale 10 μm.

OOCYTE STAGE 13 chromosome Rod accumulated at kinetochore Sentin microtubule OOCYTE STAGE 14 kinetochore with no Rod accumulated microtubule with Rod streaming Fig.62. A model of Sentin function in destabilization of kinetochore-microtubule attachments in early oocyte stages. Sentin localizes to microtubule plus ends throughout the spindle and promotes catastrophes. This leads to destabilization of kinetochore-microtubule attachments. In later stages, Sentin is transferred towards the poles. Low concentration of Sentin in the area of chromosomes enables stabilization of kinetochore-microtubule attachments.

* * * OOCYTESTAGE: EARLY 13 LATE 13 14 oocyte nurse cells area
appendages * * pre-congressed chromosomes de-congressed chromosomes re-congression and tightening of chromosomes unstable chromosome orientation stable chromosome orientation unstable kinetochore-microtubule attachments stable kinetochore-microtubule attachments * nuclear envelope kinetochore-dependent transition spindle range of time of major Augmin function in chromosome congression chromosomes crucial steps Sentin-dependent in regulation of chromosome orientation Fig.63. Model for timely participation of kinetochores, Augmin and Sentin in chromosome positioning in Drosophila oocytes. Oocytes of stages 13 to 14, defined by oocyte appearance, correspond to different maturation stage of the division apparatus. In early stage 13, nuclear envelope breaks down, chromosomes de-congress change orientation and re-congress. In the following stages, the spindle shortens and metaphase plate tightens. Kinetochores lead all three-steps of chromosome positioning : de-congression, change of orientation and re-congression. Augmin function is particularly important for limiting chromosome span in the stage 13. Sentin is crucial for orientation change in early stage 13.

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