Houliston E et al. (1994), The kinesin-related protein Eg5 associates with...

XB-ART-21080

Dev Biol 1994 Jul 01;1641:147-59. doi: 10.1006/dbio.1994.1187.

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The kinesin-related protein Eg5 associates with both interphase and spindle microtubules during Xenopus early development.

Houliston E , Le Guellec R , Kress M , Philippe M , Le Guellec K .

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We have examined the changing abundance and distribution of the kinesin-related protein Eg5 during oogenesis and early development in Xenopus laevis. Antibodies raised against proteins synthesized from parts of a novel Eg5 gene expressed in eggs were used for Western blotting and immunofluorescence. Eg5 protein was highly enriched in oocytes and eggs compared with other adult tissues. It accumulated during the latter stages of oogenesis and increased a further threefold during oocyte maturation. Its level then gradually declined during early development. In oocytes, eggs, and early embryos, Eg5 protein could be detected throughout the cytoplasm and in subcortical aggregates. Eg5 staining was found concentrated in meiotic and mitotic spindles, mainly toward the poles. Some Eg5 staining colocalized with microtubules in interphase cells, including the aligned subcortical microtubules in fertilized eggs implicated in the cortical rotation that specifies the dorsoventral axis. Interphase association of Eg5 with microtubules during early development was confirmed by copelleting the protein with microtubules from egg homogenates. In tadpoles and tissue culture cells, Eg5 colocalized with spindle microtubules throughout mitosis but not with interphase microtubules. These results suggest that the Eg5 microtubule motor may function in meiosis, mitosis, and interphase during early development.

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Species referenced: Xenopus laevis
Genes referenced: kif11

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	FIG. 1. (A) Nucleotide and predicted amino acid sequence of the second Eg5 eDNA (XLEg5K2), aligned with that of XLEg5Kl. Conserved amino acids are not indicated. (B) Diagram showing the homologies (similar/identical amino acids) in different domains of XLEg5Kl and XLEg5K2 and the organization of partial sequences expressed in bacteria. BESTFIT was used to find the best segments of similarities between the two sequences (Devereux et aL, 1984). Domain structure from Le Guellec et aL, 1991; Sawin et aL, 1992a.
	FIG. 2. Eg5 protein in Xenupu.~ oocytes, eggs, embryos, and adult tissues detected by Western blotting. In parts a-e the 135-kDa Eg5 band obtained with the anti-tail serum is shown. (a) Comparison of Eg5 levels in unfertilized eggs (UFE) and various adult tissues. (b) Eg5 levels in oocytes at successive stages of oogenesis (I-VI) with equivalent amounts of total protein loaded. (c) Same stages but with one oocyte equivalent loaded per lane. (d) Accumulation ofEg5 during maturation (hr after progesterone treatment-one oocyte equivalent per lane). In this experiment 90% of oocytes showed white maturation spots 8 hr after progesterone treatment. (e) Decline in Eg5levels during embryogenesis. One embryo equivalent from each stage indicated was loaded. (f) Full lanes containing samples from unfertilized eggs, showing the specificity of the affinity-purified anti-tail antibody and the C9 serum. The position of migration of molecular weight standards (kDa) is marked.
	FIG. 3. Meiotic spindles double stained by immunofluorescence (whole mount) with anti-tubulin (a-g) and various anti-Eg5 (a'-g') antibodies: (a') C9 serum; (b') C9 serum preabsorbed with C9 fusion protein; (c') anti-tail serum; (d') anti-tail serum preabsorbed with tail protein; (e'-g') affinity-purified anti-tail antibody. (a-e) Metaphase II spindles from unfertilized eggs; (f) a monaster found during spindle formation; and (g) a metaphase I spindle. Bar. 20 .urn for a-d, 10 .urn fore-g.
	FIG. 4. Fertilized eggs double stained with anti-tubulin (a-f) and anti-Eg5 tail (a'-f') antibodies (affinity purified in all but d'). Sections of eggs fixed during the first half of the cell cycle (0.30-0.35 NT) show sperm aster microtubules extending from the animal to the vegetal hemisphere (a) but not near the vegetal cortex (b). A portion of the heavy cytoplasmic Eg5 staining in the animal cytoplasm appears to become associated with the microtubules. The aggregates of Eg5 staining (arrow) beneath the vegetal cortex can be seen from the vegetal surface of whole eggs (c). In eggs fixed during the second half of the cell cycle (0.65-0.70 NT) a dense microtubule array with associated Eg5 staining can be seen beneath the vegetal cortex in sections (d) and from the surface of whole eggs (e. f). Y, autofluorescence from large vegetal yolk platelets. VC, vegetal cortex. Bar, 10 ,u.m.
	FIG. 5. Association of Eg5 with micro tubules in fertilized eggs during the first cell cycle. Homogenized eggs were fractionated into polymeric tubulin pellets (P, 3.5 egg equivalents per lane) and cytoplasmic supernatants (S, 1 egg equivalent per lane) at successive times during the first cell cycle. Times are expressed on a normalized time scale where 1 NT is first cleavage (I 10 min after fertilization in this experiment). Western blots using anti-Eg5 tail (135-kDa band) and anti-tubulin (55-kDa band).
	FIG. 6. Immunofluorescence with Eg5 ant ibodies in embryonic and adult cells. (a) Section of a mitotic cell from an early blastula. (b) Mitotic and interphase cells in early neurula epithelial cells (whole mount). (c) Metaphase (m), telophase (t), and interphase (i) cells in tadpole epidermis. (d) Eg5 staining enriched in a mitotic cell but not axons (n) in t he trunk of a older tadpole. (e-h) Eg5 staining in Xenopu.~ A6 cells at different mitotic stages (p, prophase; m, metaphase; a, anaphase; t, telophase; i, interphase). Embryos were double stained with anti·tubulin (a-d) and affinity-purified anti-Eg5 tail (a'-d') antibodies and A6 cells with anti-tubulin (c, g) and C9 (e', g') antibodies or Hoechs t dye (f, h) and C9 (f', h' ). Bar, 10 I'm, except for f and h (12 I'm).
	FIG. 7. Relationship of Eg5 with the ER. Double immunofluorescence with anti-BiP (a-f) and affinity-purified anti-tail (a'-f') antibodies. (a) Vegetal cortical region of a section of a fertilized egg fixed at 0.35 NT. (b) similar region at 0.65 NT. (c) Vegetal surface of whole fertilized egg fixed at 0.70 NT. (d) View from the animal pole of an unfertilized egg showing the top of the vertical meiotic spindle. (e) Section of a mitotic blastula cell. (f) Section of an interphase blastula cell. There is partial colocalization of Eg5 and ER around the microtubules of the vegetal array in fertilized eggs; however, BiP is excluded from the meiotic spindle, whereas Eg5 is concen\.rated inside it. Y, weak autofluorescence {rom large vegetal yolk platelets. VC, vegetal cortex. Bar, 10 I'm.