Fidelity of spindle assembly and function is of vital importance for the fate of resulting daughter cells. In mitosis, improper chromosome segregation often leads to cell death through mitotic catastrophe 
and has also been linked to cancer through formation of aneuploid cells 
. Anomalies during meiotic divisions of the oocyte lead to embryo aneuploidy, and can result in embryonic death or genetic disorders 
such as trisomies. The latter are often associated with tumor development 
. Several factors might lead to oocyte-related aneuploidy. They include chromosome non-disjunction in meiosis I and II (MI and MII) 
, as well as diverse factors leading to spindle morphology abnormalities. Such factors may be of a toxic chemical nature 
or age-related 
, (rev. in 
). The fact that perturbing functions of proteins other than DNA recombination factors and tubulin may lead to aneuploidy was illustrated by the interference with mitotic kinesin Eg5 in mouse oocytes 
. Although the relative contribution of the microtubule-dependent spindle abnormalities versus chromosome recombination defects to the total number of embryonic aneuploidy and failures of development events is unclear, evidence suggests that spindle assembly defects account for at least some of them 
Although meiosis and mitosis share a lot of similarities, numerous differences exist between the spindles in these types of cells and some of these differences have been already identified: (i) centrosomes are present in somatic cells and absent in oocytes; (ii) kinetochores microtubules appear very late in MI 
; (iii) Ran GTPase, crucial for spindle assembly in mitosis and MII, seems to be dispensable for MI 
Uncovering novel components of the meiotic microtubule proteome (MeMP) should not only provide clues to pathways common to both mitotic and meiotic spindle, but may reveal factors specific to microtubule-related processes in meiosis.
The spindle is often described as a dynamic assembly of chromosomes, microtubules and regulatory proteins 
. Both mitotic and meiotic spindle microtubules are stabilized and organized into a bipolar shape by a number of accessory proteins, called microtubule motors and MAPs (for “Microtubule-Associated Proteins”)  
. Motors, which include cytoplasmic dynein and members of the kinesin superfamily, use the energy of ATP hydrolysis to generate force to move along microtubules (rev. in 
. Other activities of specialized kinesin-like proteins, such as microtubule depolymerization by proteins of the kinesin 13 family, also require ATP hydrolysis 
. Major functions of motors in the meiotic spindle include connecting the plus ends of microtubules, positioning the chromosomes at the equator of the spindle and focusing the poles, which is particularly important in the absence of centrosomes. MAPs are usually defined as proteins which bind in vivo
and in vitro
to microtubules, co-localize with microtubules in the cell 
and affect microtubule polymerization dynamics 
. During oogenesis, MAPs are believed to stabilize microtubules preferentially close to condensed chromosomes, favoring spindle assembly around DNA. Both MAPs and motors can tether other proteins and organelles to microtubules in vivo 
. In vitro
, the association of motors with microtubules is ATP-sensitive, while MAPs can be eluted by salt. In this way, individual proteins and large protein complexes can be purified on microtubules in amounts sufficient for biochemical analysis of the microtubule-associated proteome 
Why use Xenopus
eggs to study MeMP? Several recent proteomic studies targeted proteins associated with the microtubule cytoskeleton: centrosome from human lymphoblastic cell line KE37 
, mitotic spindle from HeLa cells 
, midbody in hamster CHO cells 
, and microtubule asters in extracts from mitotic HeLa cells 
. Using mammalian oocytes to study MeMP would be problematic for availability reasons. On the contrary, Xenopus laevis
oocytes are easily procured in large quantities and have been long used to study meiosis thanks to the conservation of spindle assembly pathways between vertebrate species. Extracts prepared from unfertilized Xenopus
represent an abundant source of cytoskeletal and cell cycle proteins. Indeed, during the first 12 divisions after fertilization very little protein translation occurs, with the egg providing most of the proteins needed for these rapid divisions 
. The uniqueness of Xenopus
egg extract experimental system lies in the possibility to study many aspects of the spindle assembly in a cell-membrane-free context. Because of this, numerous spindle-related studies have been realized in this system and both the methodology and tools are available for the investigation of novel spindle components. The extracts are usually prepared from eggs arrested in the metaphase of MII (“cytostatic factor-arrested”) and are immediately competent for spindle assembly experiments. We therefore chose Xenopus
egg extracts as a source of meiotic proteins.
In a recent proof-of-the-principle study we used mass spectrometry and sequence-similarity searches to identify a subset of 41 components of the Xenopus
. Building up on these results we set out to decipher the first comprehensive MeMP. Using similar purification strategy together with LC-MS/MS analysis, combined with conventional (Mascot) and sequence-similarity searches 
we established a catalogue of the proteins bound to microtubules in Xenopus
egg extracts comprising 318 individual entries. Many of these proteins were either previously uncharacterized or their association with meiotic/mitotic spindle was not reported.
We next assembled the identified proteins into the first literature-curated network of the Meiotic Microtubule-associated Protein Interaction Network (MeMPIN), to show interactions of proteins complexes and individual proteins with microtubules. The network also highlighted a number of proteins and their complexes absent from the mitotic spindle proteome (MSP), suggesting that some of them may have a specific microtubule-related role during oogenesis. Furthermore, we compiled alternative networks, which include high and low-confidence data from interaction databases and yeast-two-hybrid studies on functional and structural orthologues of Xenopus proteins. All three Cytoscape-based versions of MeMPIN are interactive and available online.
Further, we evaluated the localization of nine proteins, all of which were not previously reported as components of MSP. For this, we used expression of their GFP-tagged cDNA in cultured cells and addition of GFP-tagged proteins to egg extracts. In this way we showed that seven out of nine new proteins (Mgc68500, Loc398535, Nif3l1bp1/THOC7, TSGA14/CEP41, LSM14A/RAP55, Mgc80361 and Mgc81475) localize to the spindle. Probing the functional role of these proteins, we used siRNA to interfere with the expression of their human orthologues in HeLa cells and showed that depletion of huMGC81475 destabilizes spindle assembly, arresting cells in mitosis.