David L. Gard
Professor of biology
B.S. California Institute of Technology
Ph.D. California Institute of Technology
Research
Like most eukaryotic cells, oocytes and embryos of Xenopus laevis (the African clawed toad) contain three systems of cytoplasmic filaments: microtubules, actin filaments (F-actin), and intermediate filaments. This "cytoskeleton" forms a complex and dynamic network that has been postulated to play a number of critical roles, including the establishment and maintenance of cell and embryonic polarity, and meiotic and mitotic cell divisions.
We are trying to understand the regulation and role of microtubules during oogenesis and early development in the African clawed toad, Xenopus laevis . Currently, we are focusing our attention on two MT associated proteins, XMAP215 and XMAP230, found in Xenopus oocytes, eggs, and early embryos. These XMAPs have very different effects on MT assembly in vitro : XMAP230 promotes assembly by stabilizing MTs, while XMAP215 promotes the assembly of long, but highly dynamic, MTs. Confocal immunofluorescence microscopy reveals that both XMAPs are associated with spindle MTs during mitosis. XMAP 230 also associates with interphase MTs, including the novel network of subcortical MTs required for formation of the D-V axis in early Xenopus embryos. Their distinct distributions, differing effects on MT assembly in vitro , and the effects of morpholino antisense oligonucleotides targeting XMAP215 or XMAP230 mRNA suggest that XMAP215 and XMAP230 play very different roles in regulating MT assembly and organization in vivo . The studies carried out in eggs and early embryos are being carried out in parallel with complimentary studies of the roles of XMAP215 and XMAP230 in regulating microtubule dynamics and organization in cultured Xenopus XTC cells.
Finally, recent evidence indicates that XMAP215 is a member of the ancient MAP215/Dis1 family of microtubule-associated proteins that traces its roots to the very origin of eukaryotic cells. Studies conducted in a variety of model organisms indicate that MAP215/Dis1 proteins play critical roles in the assembly and/or function of mitotic spindles and eukaryotic cell division. We are using a variety of approaches, including bioinformatics and molecular cloning of MAP215/Dis1 proteins in non-model organisms, to further study the evolution of the MAP215/Dis1 family in relationship to their structure and function.
XMAP215 is associated with microtubules of the mitotic spindle in Xenopus blastulae (green channel=XMAP215; red channel=tubulin; blue channel=DNA).
References
1. Gard DL, Becker BE, Romney SJ (2004) MAPping the eukaryotic tree of life: the structure, function, and evolution of the MAP215/Dis1 family of microtubule-associated proteins. International Reviews of Cytology 239:179-272
2. Becker BE, Romney SJ, Gard DL (2003) XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes . Developmental Biology 261(2):488-505
3. Becker B, Gard DL (2000) Multiple isoforms of XMAP215 are expressed during oogenesis and early development in Xenopus laevis . Cell Motility and the Cytoskeleton 47:282-295
4. Cha B-J, Cassimeris L, Gard DL (1999) XMAP230 is required for normal spindle assembly in vivo and in vitro. J. Cell Sci. J. Cell Science 112:4337-4346
5. Pfeiffer DC, Gard DL (1999) Microtubules in Xenopus oocytes are oriented with their minus-ends towards the cortex. Cell Motil and Cytoskeleton 44:34-43
6. Gard DL (1999) Confocal microscopy and 3-D reconstruction of the cytoskeleton of Xenopus oocytes. Micros. Res. Tech. 44:388-414
7. Cha B-J, Gard DL (1999) XMAP230 is required for the assembly and organization of cortical microtubules in fertilized Xenopus eggs. Devel. Biol. 205:275-286
8. Cha B-J, Error B, Gard DL (1998) XMAP230 is required for the assembly and organization of acetylated microtubules in Xenopus oocytes and eggs. J. Cell Sci. 111:2315-2327
9. Charrasse S, Schroeder M, Gauthier-Rouviere C, Ango F, Cassimeris L, Gard D, Larroque C (1998) The human TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215. J. Cell Sci. 111:1371-1383
10. Gard DL, Cha B-J, King E (1997) The organization and animal-vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon F-actin and Microtubules. Developmental Biology 184:95-114 (cover)
11. Gard DL, Affleck D, Error B (1995) Microtubule organization, acetylation, and nucleation in Xenopus laevis oocytes: II. A developmental transition in microtubule organization during early diplotene. Devel. Biol. 168:189-201
