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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 the mouse ASAP. (A) Characterization of anti-mouse ASAP antibodies by immunoblotting. Non-transfected (NT) or transfected U2-OS (with a human ASAP siRNA) or NIH3T3 (with EYFP or EYFP-mASAP) cells were blotted first with an anti-GFP (left), then with the anti-mASAP (right). The specificity of the affinity-purified anti-mASAP antibody was assessed by using cell extracts from NIH3T3 and U2-OS (right), and compared to the profile observed 1) after ASAP-depletion by siRNA in the U2-OS cells (right) and 2) with the anti-GFP antibody against EYFP-mASAP transfected NIH3T3 cells (left). * : non specific bands observed with the α-GFP and that still show up in the α-mASAP blot since the same membrane was used. The same blot was finally probed with an anti-α-tubulin to demonstrate equal loading (bottom). The sizes of molecular weight marker are shown on the left. (B) Subcellular localization of endogenous mASAP during the cell cycle. Exponentially growing NIH3T3 cells were fixed with PFA and processed for immunofluorescence with the affinity-purified antibody against mASAP (green), and an antibody against α-tubulin (red). DNA was stained with Hoechst dye 33258 (blue). (C) Overexpression of mASAP leads to MTs bundles and monopolar spindle formation in NIH3T3 cells. Forty-eight hours after transfection with 2 μg of EYFP-mASAP, cells were fixed with PFA and stained with an anti-α-tubulin (1) or an anti-GT335 (2) (red) and Hoechst 33258 (DNA). Insets show a higher magnification of GT-335 foci. EYFP signals are in green. (Bars, 10 μm).
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Figure 7: Characterization of the mouse ASAP. (A) Characterization of anti-mouse ASAP antibodies by immunoblotting. Non-transfected (NT) or transfected U2-OS (with a human ASAP siRNA) or NIH3T3 (with EYFP or EYFP-mASAP) cells were blotted first with an anti-GFP (left), then with the anti-mASAP (right). The specificity of the affinity-purified anti-mASAP antibody was assessed by using cell extracts from NIH3T3 and U2-OS (right), and compared to the profile observed 1) after ASAP-depletion by siRNA in the U2-OS cells (right) and 2) with the anti-GFP antibody against EYFP-mASAP transfected NIH3T3 cells (left). * : non specific bands observed with the α-GFP and that still show up in the α-mASAP blot since the same membrane was used. The same blot was finally probed with an anti-α-tubulin to demonstrate equal loading (bottom). The sizes of molecular weight marker are shown on the left. (B) Subcellular localization of endogenous mASAP during the cell cycle. Exponentially growing NIH3T3 cells were fixed with PFA and processed for immunofluorescence with the affinity-purified antibody against mASAP (green), and an antibody against α-tubulin (red). DNA was stained with Hoechst dye 33258 (blue). (C) Overexpression of mASAP leads to MTs bundles and monopolar spindle formation in NIH3T3 cells. Forty-eight hours after transfection with 2 μg of EYFP-mASAP, cells were fixed with PFA and stained with an anti-α-tubulin (1) or an anti-GT335 (2) (red) and Hoechst 33258 (DNA). Insets show a higher magnification of GT-335 foci. EYFP signals are in green. (Bars, 10 μm).

Mentions: We first investigated if mASAP had the same endogenous and overexpression patterns as its human counterpart. We raised polyclonal antibodies against the full-length protein. Affinity-purified antibodies recognized an endogenous single protein band of ~110 kDa in NIH3T3 and U2-OS cells, as observed with human ASAP (Fig. 7A, right). The specificity of the antibodies was assessed by comparing these profiles to that observed with the anti-GFP antibody against EYFP-mASAP transfected cells (Fig. 7A, left), and to ASAP-depleted U2-OS cells by RNA interference (Fig. 7A, right). The endogenous signal disappeared in siRNA-transfected cells and the anti-mASAP recognized the highest molecular form of EYFP-mASAP (~137 KDa, highlighted by the EYFP-antibody) in the corresponding NIH3T3-transfected cells. It is noteworthy that, as suspected by sequence comparison, the mouse antibody is able to recognize the human protein. Unfortunately, as observed for the human antibodies, the two antibodies generated against mASAP gave a significant background in immunofluorescence studies. The same ASAP localization was observed using several fixation methods in U-2 OS (not shown) and NIH3T3 cell lines. We observed an identical subcellular localization between endogenous mASAP and human ASAP. Mouse ASAP co-localizes with the MT network in interphase. During mitosis it first relocates to the poles at the astral MTs and the spindle during prometaphase, metaphase and early anaphase, and then to centrosomal MT asters and the central spindle during anaphase and early telophase. During late cytokinesis mASAP colocalizes with the midzone MT bundles when it is also located on MTs nucleated from centrosomes (Fig. 7B).


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 the mouse ASAP. (A) Characterization of anti-mouse ASAP antibodies by immunoblotting. Non-transfected (NT) or transfected U2-OS (with a human ASAP siRNA) or NIH3T3 (with EYFP or EYFP-mASAP) cells were blotted first with an anti-GFP (left), then with the anti-mASAP (right). The specificity of the affinity-purified anti-mASAP antibody was assessed by using cell extracts from NIH3T3 and U2-OS (right), and compared to the profile observed 1) after ASAP-depletion by siRNA in the U2-OS cells (right) and 2) with the anti-GFP antibody against EYFP-mASAP transfected NIH3T3 cells (left). * : non specific bands observed with the α-GFP and that still show up in the α-mASAP blot since the same membrane was used. The same blot was finally probed with an anti-α-tubulin to demonstrate equal loading (bottom). The sizes of molecular weight marker are shown on the left. (B) Subcellular localization of endogenous mASAP during the cell cycle. Exponentially growing NIH3T3 cells were fixed with PFA and processed for immunofluorescence with the affinity-purified antibody against mASAP (green), and an antibody against α-tubulin (red). DNA was stained with Hoechst dye 33258 (blue). (C) Overexpression of mASAP leads to MTs bundles and monopolar spindle formation in NIH3T3 cells. Forty-eight hours after transfection with 2 μg of EYFP-mASAP, cells were fixed with PFA and stained with an anti-α-tubulin (1) or an anti-GT335 (2) (red) and Hoechst 33258 (DNA). Insets show a higher magnification of GT-335 foci. EYFP signals are in green. (Bars, 10 μm).
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Figure 7: Characterization of the mouse ASAP. (A) Characterization of anti-mouse ASAP antibodies by immunoblotting. Non-transfected (NT) or transfected U2-OS (with a human ASAP siRNA) or NIH3T3 (with EYFP or EYFP-mASAP) cells were blotted first with an anti-GFP (left), then with the anti-mASAP (right). The specificity of the affinity-purified anti-mASAP antibody was assessed by using cell extracts from NIH3T3 and U2-OS (right), and compared to the profile observed 1) after ASAP-depletion by siRNA in the U2-OS cells (right) and 2) with the anti-GFP antibody against EYFP-mASAP transfected NIH3T3 cells (left). * : non specific bands observed with the α-GFP and that still show up in the α-mASAP blot since the same membrane was used. The same blot was finally probed with an anti-α-tubulin to demonstrate equal loading (bottom). The sizes of molecular weight marker are shown on the left. (B) Subcellular localization of endogenous mASAP during the cell cycle. Exponentially growing NIH3T3 cells were fixed with PFA and processed for immunofluorescence with the affinity-purified antibody against mASAP (green), and an antibody against α-tubulin (red). DNA was stained with Hoechst dye 33258 (blue). (C) Overexpression of mASAP leads to MTs bundles and monopolar spindle formation in NIH3T3 cells. Forty-eight hours after transfection with 2 μg of EYFP-mASAP, cells were fixed with PFA and stained with an anti-α-tubulin (1) or an anti-GT335 (2) (red) and Hoechst 33258 (DNA). Insets show a higher magnification of GT-335 foci. EYFP signals are in green. (Bars, 10 μm).
Mentions: We first investigated if mASAP had the same endogenous and overexpression patterns as its human counterpart. We raised polyclonal antibodies against the full-length protein. Affinity-purified antibodies recognized an endogenous single protein band of ~110 kDa in NIH3T3 and U2-OS cells, as observed with human ASAP (Fig. 7A, right). The specificity of the antibodies was assessed by comparing these profiles to that observed with the anti-GFP antibody against EYFP-mASAP transfected cells (Fig. 7A, left), and to ASAP-depleted U2-OS cells by RNA interference (Fig. 7A, right). The endogenous signal disappeared in siRNA-transfected cells and the anti-mASAP recognized the highest molecular form of EYFP-mASAP (~137 KDa, highlighted by the EYFP-antibody) in the corresponding NIH3T3-transfected cells. It is noteworthy that, as suspected by sequence comparison, the mouse antibody is able to recognize the human protein. Unfortunately, as observed for the human antibodies, the two antibodies generated against mASAP gave a significant background in immunofluorescence studies. The same ASAP localization was observed using several fixation methods in U-2 OS (not shown) and NIH3T3 cell lines. We observed an identical subcellular localization between endogenous mASAP and human ASAP. Mouse ASAP co-localizes with the MT network in interphase. During mitosis it first relocates to the poles at the astral MTs and the spindle during prometaphase, metaphase and early anaphase, and then to centrosomal MT asters and the central spindle during anaphase and early telophase. During late cytokinesis mASAP colocalizes with the midzone MT bundles when it is also located on MTs nucleated from centrosomes (Fig. 7B).

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