Further Characterization of a Novel Membrane-Microtubule Binding Protein, CLIPR76

Microtubules are essential to cellular organization and are thus regulated by many proteins. One family of these regulatory proteins is known as the CLIPR (CLIP-170 Related) family, so-called because its members contain conserved CAP-GLY domains originally found in CLIP-170. This thesis describes the characterization of an alternatively spliced member of this family, CLIPR76, by a combination of cell biological, biochemical, and bioinformatic approaches.

CLIPR76-4, the smallest human splice isoform, contains a hydrophobic C-terminus that drives ER localization and aligns with the C-terminal Golgi-localizing domain of the CLIPR76 relative CLIPR-59. The mechanism of CLIPR76-4 membrane localization was investigated by mutating a key conserved cysteine necessary for CLIPR-59 localization. Cells transfected with GFP-fusion mutant CLIPR76-4 were visualized by immunofluorescence microscopy displayed unperturbed ER localization. This indicates that CLIPR76-4 and CLIPR-59 target their membranes via distinct mechanisms.

The nature of the CLIPR76-microtubule interaction was then investigated. Microtubule cosedimentation assays with purified proteins showed that two of the CLIPR76 CAP-Glys, including one common to all isoforms, bind MTs with affinities in the 1åµM range and also promote MT polymerization. These observations demonstrate that CLIPR-76 proteins act on MTs directly and do so through their conserved CAP-GLY domains. While the MT-binding ability of the CLIPR76 CAP-GLY domains is similar to that of the CLIP-170 CAP-Gly domains, cosedimentation assays in the presence of EB1 showed that CLIPR76 lacks that ability to bind EB1.

Bioinformatics was then used to identify aspects of the CAP-GLY structures that could mediate these different activities. Multiple sequence alignments of several CAP-Gly domains were generated and then mapped onto their corresponding 3D structures. There is a large conserved patch on all of the CLIPR76 CAP-Gly domains, corresponding to residues that may mediate MT-CAP-Gly interactions. A comparison of the two CAP-Gly domains of CLIP-170 revealed a region of non-conservation which could explain the two CAP-Glys' different affinities for EB1. These regions of non-conservation remain important targets for future mutagenesis studies.

Finally, using a K-12 outreach opportunity, a brief survey was developed to rate students' attitudes towards science and showed that interactions with scientists and inquiry based projects significantly improved student attitudes towards science.