David L. Gard

Professor of Biology

David Gard

B.S. California Institute of Technology

Ph.D. California Institute of Technology

Research

References

gard@biology.utah.edu

David Gard's Lab Page

David Gard's PubMed Literature Search

Molecular Biology Program

Biological Chemistry Program

Cytoskeletal Function & Development

Research

Research in my laboratory focuses on the structure, function, and evolution of XMAP215, a high molecular weight microtubule-associated protein (MAP) first isolated from eggs of the African clawed frog, Xenopus laevis. Recent studies reveal that XMAP215 is a member of the MAP215/Dis1 family of MAPs. Genes and/or transcripts encoding MAP215/Dis1 orthologs have now been identified in >250 species spanning the eukaryotic branch of the tree of life, indicating that the MAP215/Dis1 traces its roots to the very origin of eukaryotic cells. Studies conducted in a variety of model organisms, including Xenopus, indicate that MAP215/Dis1 proteins play critical roles in regulating the assembly and/or function of microtubules and mitotic spindles, key components of the division apparatus of eukaryotic cells.

In our examination of XMAP215 expression during Xenopus development, we found that two mRNAs, encoding distinct isoforms of XMAP215 protein, are produced by alternative splicing of the XMAP215 transcript. The two XMAP215 proteins differ in the presence (XMAP215M) or absence (XMAP215Z) of a 36 amino acid sequence called insert 2 (ins2). XMAP215M (w/ ins2) is expressed during oogenesis and present through early gastrulation, while XMAP215Z (w/o ins2) is expressed from gastrulation onward. Ins2 includes a predicted target for the cell cycle kinase CDK1, suggesting that it might play a role in regulating XMAP215 activity during the rapid cells cycles of early Xenopus development.

Surprisingly, the single exon encoding ins2 is absent from the MAP215 genes of ray-finned fishes (zebra fish, stickleback, and pufferfish) and mammals (monotremes, marsupials, and eutherians). Initially, this suggested that ins2 might be a feature peculiar to amphibians. However, the discovery of a sequence encoding ins2 in the MAP215 gene of chickens, and confirmation that the chicken ins2 exon is expressed in mRNA, indicates that splicing of MAP215 transcripts to produce M- and Z-forms of MAP215 originated in a common ancestor of terrestrial vertebrates, and has secondarily been lost in mammals. To test this hypothesis, we are amplifying and sequencing regions spanning ins2 in MAP215 transcripts and genes from additional vertebrate species. We have now sequenced the relevant regions of MAP215 genes/transcripts from a dozen vertebrate species, from sharks to reptiles. Our results suggest that the ins2 exon first arose in MAP215 genes of sarcopterygians (lobe-finned fish and terrestrial vertebrates) subsequent to their divergence from actinopterygians (ray-finned fish).

We are also studying the regulation of XMAP215 function by phosphorylation. Results from previous studies revealed that XMAP215 is hyperphosphorylated in M-phase, and that phosphorylation by CDK1 modulates XMAP215 activity in vitro. However, the sequence of XMAP215M includes 270 phosphorylatable amino acids (Ser/Thr/Tyr). Nearly 100 of these are predicted by surface probability and sequence comparisons to be phosphorylated, providing a myriad of possibilities for fine-tuning XMAP215 function in vivo. These “predicted” sites are not randomly distributed in the primary sequence, but group into clusters associated with mapped functional domains, and include target sites for cyclin-dependent kinases, Aurora A, MARK, and other kinases known/thought to regulate microtubule functions. In preliminary studies, we have used mass spectroscopy to identify and map 22 phosphorylation sites in XMAP215M isolated from unfertilized Xenopus eggs. Suprisingly, fully half (11/22) of these sites fall within the 36 amino acid insert 2! We are currently generating GFP-tagged XMAP215 bearing site-directed mutations in these phosphorylation sites to investigate the role of these sites in regulating the association of XMAP215 with microtubules and microtubule-organizing centers in Xenopus tissue culture (XTC) cells, oocytes, and embryos.

Gard Figure
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

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Last Updated: 11/7/16