Xenopus meiotic microtubule-associated interactome.
In metazoan oocytes the assembly of a microtubule-based spindle depends on the activity of a large number of accessory non-tubulin proteins, many of which remain unknown. In this work we isolated the microtubule-bound proteins from Xenopus eggs. Using mass spectrometry we identified 318 proteins, only 43 of which are known to bind microtubules. To integrate our results, we compiled for the first time a network of the meiotic microtubule-related interactome. The map reveals numerous interactions between spindle microtubules and the newly identified non-tubulin spindle components and highlights proteins absent from the mitotic spindle proteome. To validate newly identified spindle components, we expressed as GFP-fusions nine proteins identified by us and for first time demonstrated that Mgc68500, Loc398535, Nif3l1bp1/THOC7, LSM14A/RAP55A, TSGA14/CEP41, Mgc80361 and Mgc81475 are associated with spindles in egg extracts or in somatic cells. Furthermore, we showed that transfection of HeLa cells with siRNAs, corresponding to the human orthologue of Mgc81475 dramatically perturbs spindle formation in HeLa cells. These results show that our approach to the identification of the Xenopus microtubule-associated proteome yielded bona fide factors with a role in spindle assembly.
PubMed ID: 20174651
PMC ID: PMC2822853
Article link: PLoS One.
Genes referenced: actl6a cep41 csnk1g2 itih3 lrrfip2 lsm14a mst1 plec thoc7
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|Figure 1. Isolation of microtubule-binding proteins from Xenopus egg extracts.A. Scheme of the protein purification using precipitation on Taxol-stabilized microtubules. B. Coomassie-stained SDS gradient gels showing protein fractions resolved on 5–40% sucrose gradients. Upper and lower gels show the ATP and NaCl elution fractions, respectively. Only fractions 1–15 are shown from the total of 20. PAA- polyacrylamide. kD- kilodaltons.|
|Figure 2. Meiotic microtubule-associated protein interaction network.Map contains 218 proteins, 75% of all characterized proteins identified in our study, and 51 complexes. Note that EF1-α, CKI-α, Need1, tubulin ε and Plectin 1 are shown as MAPs because they can bind to microtubules directly. The identified by us spindle components (Mgc68500, TSGA14/CEP41, LSM14A/RAP55A, Mgc80361, Mgc81475; see Figure 3) are linked to the microtubule using a dashed line, since their binding may be indirect. Similarly, a dashed line is used to show the interaction of Loc398535/LRRFIP2 with actin.|
|Figure 3. Localization of GFP-tagged candidate proteins at egg extract spindles.A. Spindles assembled in egg extracts in the presence of GFP-tagged candidate proteins. Size bar – 20 µm. Overlay column shows red (tubulin), green (GFP) and blue (DNA) signals. B. Relative intensities of the GFP fluorescence of the spindles assembled in the presence of nine GFP-tagged candidate proteins, normalized to the Rhodamine-tubulin signal. Values represent average from at least 10 spindles per sample. The ratio of GFP signal to tubulin fluorescence intensity for GFP alone was considered as 1. Error bars show s.e.m.|
|Figure 4. huMgc81475 depletion perturbs spindle assembly.A. Immunofluorescence on fixed HeLa cells after transfection with control and huMgc81475 siRNAs. Cells were stained with anti-phospho-histone H3 (H3P) and anti-tubulin antibodies. Bottom panel shows three representative phenotypes in cells treated with huMgc81475 siRNAs #3 and 4. Size bar – 10 µm. B. Quantification of phenotypes observed as a result of the huMgc81475 knockdown. Morphological stages of mitosis were evaluated for control (white bars; N = 135) and huMgc81475 siRNA-treated H3P-positive cells (black bars; N = 254). C. Percent of mitotic cells with unaligned chromosomes in control- and huMgc81475 siRNA-treated cells. Only mitotic cells with bipolar structures were evaluated.|
|Figure 5. Proteins common to meiosis microtubule-associated protein interactome and mitotic spindle proteome.Same as Figure 2 with proteins common to both MeMP and MSP shown in light color. Note that some of the 90 common proteins are not shown on the network (see Table S3 legend).|