Cytoplasmic Linker Proteins: Keeping in Shape by Regulating the Cytoskeleton

Abstract

Microtubules are an essential component of the eukaryotic cytoskeleton. Proper functioning of the microtubular network depends highly on its dynamic properties, with frequent transitions from phases of prolonged growth to rapid shrinkage (catastrophe) and vice versa (rescue). Several proteins have been shown to bind to microtubules, some of them, the so-called plus-end-tracking proteins (+TIPs), binding specifically to the growing ends of microtubules. This thesis describes the functional analysis of two +TIPs, the Cytoplasmic Linker Proteins CLIP-115 and CLIP-170. These proteins are structurally highly similar resulting in a high level of redundancy. Although single CLIP-115 or CLIP-170 knockout mice both have characteristic features, no large abnormalities were found in fibroblasts derived from them. To learn more about the CLIPs and their redundant functions, CLIP double knockout (DKO) mice were generated. The dynamic properties of the microtubules in ce! lls derived from these mice were profoundly altered with a five-fold decrease in rescue-frequency. In addition, the microtubule network was changed notably, with large microtubule-free areas in which proteins aggregates were found. These aggregates contained the microtubule motor-protein cytoplasmic dynein and several components of the dynactin complex. Furthermore DKO fibroblasts spread abnormally, resulting in a two-fold increase of the surface area in attached cells compared to wild type cells. The mice deficient for both CLIP-115 and CLIP-170 have an abnormal posture and show hind limb clasping. Although they perform poorly on certain behavioral and strength-related tasks, they show a surprising age-related improvement of motor coordination. At older ages, certain tasks start to deteriorate again. Using magnetic resonance imaging (MRI) and histological analysis a progressive form of hydrocephalus is detected. In younger mice, an accumulation of cerebellar neurons is see! n in other parts of the brain, indicating a defect in neuronal migrati on. Taken together, the results described in this thesis confirm that CLIP-115 and CLIP-170 act as rescue factors, but that they also have important functions in regulating cell spreading and neuronal migration