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Gene organization, evolution and expression of the microtubule-associated protein ASAP (MAP9).

Venoux M, Delmouly K, Milhavet O, Vidal-Eychenié S, Giorgi D, Rouquier S - BMC Genomics (2008)

Bottom Line: The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a approximately110 KDa protein.The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent.It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Groupe Microtubules et Cycle Cellulaire, Institut de Génétique Humaine, CNRS UPR 1142, rue de cardonille, 34396 Montpellier cédex 5, France. Magali.Venoux@igh.cnrs.fr

ABSTRACT

Background: ASAP is a newly characterized microtubule-associated protein (MAP) essential for proper cell-cycling. We have previously shown that expression deregulation of human ASAP results in profound defects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as the domain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissue expression characterized and the overexpression profile analyzed.

Results: Bona fide ASAP orthologs are found in vertebrates with more distantly related potential orthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenic chromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a approximately110 KDa protein. The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of approximately 42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphase and to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defects similar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereas in culture neurons ASAP localized to the cell body and growing neurites.

Conclusion: The conservation of ASAP indicated in our results reflects an essential function in vertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to study the function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissues rich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and represents a key factor of mitosis with possible involvement in other cell cycle processes. It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement in neuron dynamics also highlights ASAP as a candidate target in neurodegenerative diseases.

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Characterization of Xenopus ASAP. Asynchronous XL2 cells were transfected with the EYFP-X-ASAP cDNA and analyzed (A) by immunoblot using an anti-GFP antibody or (B) by immunofluorescence. Cells were fixed in PAF/MTSB and co-stained with the anti α-tubulin (red) and Hoechst 33258 (blue).
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Figure 11: Characterization of Xenopus ASAP. Asynchronous XL2 cells were transfected with the EYFP-X-ASAP cDNA and analyzed (A) by immunoblot using an anti-GFP antibody or (B) by immunofluorescence. Cells were fixed in PAF/MTSB and co-stained with the anti α-tubulin (red) and Hoechst 33258 (blue).

Mentions: In order to develop Xenopus as a model system, we PCR-screened a Xenopus tadpole stage 24 library using primers derived from EST databases. Only one partial cDNA clone containing the N-terminal part of the sequence was isolated. The full-length clone was PCR-reconstructed using an overlapping cDNA found in the database (see methods). Given the sequence divergence between human ASAP and X-ASAP, we overexpressed the full-length EYFP-X-ASAP in Xenopus XL2 cells to observe the subcellular localization. Western blot analysis revealed an expression of EYFP constructs at ~120 kDa, a molecular weight slightly lower than that observed for hASAP or mASAP (Fig. 11A). As observed for mammalian ASAP, overexpressed X-ASAP colocalized with interphasic and mitotic MTS, showing the same localization as both mASAP and hASAP (Fig. 11B). However, we observed neither bundles nor monopolar spindles. Since the transfection level was very low, it was quite difficult to observe transfected mitosis. This may be accounted for by abnormal mitotic cells having undergone cell death.


Gene organization, evolution and expression of the microtubule-associated protein ASAP (MAP9).

Venoux M, Delmouly K, Milhavet O, Vidal-Eychenié S, Giorgi D, Rouquier S - BMC Genomics (2008)

Characterization of Xenopus ASAP. Asynchronous XL2 cells were transfected with the EYFP-X-ASAP cDNA and analyzed (A) by immunoblot using an anti-GFP antibody or (B) by immunofluorescence. Cells were fixed in PAF/MTSB and co-stained with the anti α-tubulin (red) and Hoechst 33258 (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551623&req=5

Figure 11: Characterization of Xenopus ASAP. Asynchronous XL2 cells were transfected with the EYFP-X-ASAP cDNA and analyzed (A) by immunoblot using an anti-GFP antibody or (B) by immunofluorescence. Cells were fixed in PAF/MTSB and co-stained with the anti α-tubulin (red) and Hoechst 33258 (blue).
Mentions: In order to develop Xenopus as a model system, we PCR-screened a Xenopus tadpole stage 24 library using primers derived from EST databases. Only one partial cDNA clone containing the N-terminal part of the sequence was isolated. The full-length clone was PCR-reconstructed using an overlapping cDNA found in the database (see methods). Given the sequence divergence between human ASAP and X-ASAP, we overexpressed the full-length EYFP-X-ASAP in Xenopus XL2 cells to observe the subcellular localization. Western blot analysis revealed an expression of EYFP constructs at ~120 kDa, a molecular weight slightly lower than that observed for hASAP or mASAP (Fig. 11A). As observed for mammalian ASAP, overexpressed X-ASAP colocalized with interphasic and mitotic MTS, showing the same localization as both mASAP and hASAP (Fig. 11B). However, we observed neither bundles nor monopolar spindles. Since the transfection level was very low, it was quite difficult to observe transfected mitosis. This may be accounted for by abnormal mitotic cells having undergone cell death.

Bottom Line: The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a approximately110 KDa protein.The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent.It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Groupe Microtubules et Cycle Cellulaire, Institut de Génétique Humaine, CNRS UPR 1142, rue de cardonille, 34396 Montpellier cédex 5, France. Magali.Venoux@igh.cnrs.fr

ABSTRACT

Background: ASAP is a newly characterized microtubule-associated protein (MAP) essential for proper cell-cycling. We have previously shown that expression deregulation of human ASAP results in profound defects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as the domain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissue expression characterized and the overexpression profile analyzed.

Results: Bona fide ASAP orthologs are found in vertebrates with more distantly related potential orthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenic chromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a approximately110 KDa protein. The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of approximately 42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphase and to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defects similar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereas in culture neurons ASAP localized to the cell body and growing neurites.

Conclusion: The conservation of ASAP indicated in our results reflects an essential function in vertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to study the function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissues rich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and represents a key factor of mitosis with possible involvement in other cell cycle processes. It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement in neuron dynamics also highlights ASAP as a candidate target in neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus