CLIPs: linking neuronal microtubule behaviour to disorders of the brain

Microtubules (MTs) form a dynamic cytoskeletal network that is essential for many processes, including mitosis, cell shape and structure, cell attachment and differentiation. A properly organized MT network is important for neurons for additional reasons: 1) neuronal migration critically depends on forces exerted on the MT network; 2) neurons are very long cells and the only efficient means for cellular constituents to reach neuronal extremities is by MT-based transport. The MT network is organized and maintained by MT-associated proteins. Key to this regulation are the MT “plus-end tracking proteins”, or +TIPs, which are characterized by a specific association with the ends of growing MTs. Fluorescently labelled +TIPs are widely used to mark growing MTs. We were the first to apply this technique in neurons and we documented lower MT growth rates and altered +TIP staining patterns in neurons compared to non-neuronal cells. Using newly developed tools we recently found evidence for altered dynamic behaviour of neuronal MTs. A first aim of this proposal is to examine the molecular basis underlying these differences. We will investigate MT structure and dynamics in neurons using live cell imaging with fluorescent +TIPs, and electron microscopy (EM). We will also address MT nucleation in neurons. Both novel and established fluorescence-based and biophysical methods will be used. For EM studies we have developed special procedures to preserve MT integrity. Both 2D and 3D methods will be used to view MTs and their ends. The cytoplasmic linker proteins CLIP-115 and CLIP-170 are two highly related +TIPs, with redundant roles as MT rescue factors in non-neuronal cells. To investigate CLIP function we generated single and double knockout mice, fibroblasts and neurons. Our data show that CLIPs are non-redundant in neurons. The second aim of this proposal is to examine the molecular basis for this specificity. The same approaches described above for wild type neurons will be employed to investigate MT dynamics, vesicular transport and neuronal morphology in CLIP-deficient neurons. Furthermore, we will examine the hypothesis that CLIPs act as a “landing platform” on plus-ends for other proteins. In man ~25 genes on chromosome 7q11 (including CLIP2 which encodes CLIP-115) are hemizygously deleted in patients with Williams Syndrome (WS), a neurodevelopmental disorder. Animals lacking CLIP-115 mimic aspects of WS. In CLIP-115 and -170 double knockout (DKO) mice we observe a more severe phenotype: paraventricular heterotopia and a progressive form of hydrocephalus. To examine the hypothesis that these symptoms are caused by defects in neuronal migration we will perform a spatio-temporal analysis in CLIP DKO mice using standard immunocytochemistry and novel 3D microscopy approaches, determine the onset of hydrocephalus and neuronal migration defects, and link these data to CLIP expression in the developing brain. CLIPs have been shown to interact with proteins involved in neuronal migration, i.e. LIS1, IQGAP1, and alpha-tubulin. We will map interaction domains in detail and generate subtle point mutations in the CLIPs, that abrogate specific interactions. Mutants will be examined for their ability to rescue DKO neurons. Using a biotin-streptavidin pull down approach and mass spectrometry analysis we will identify neuron-specific interaction partners of the CLIPs and of wild type and mutant alpha-tubulin. This will link CLIP function to neuronal MT behaviour and disorders of the brain. Interactions will be verified using standard cell biology approaches and functions of new partners will be addressed. Developmental and neurodegenerative disorders of the human brain have been linked to a dysfunctioning MT cytoskeleton. An optimal understanding of the molecular mechanisms involved requires fundamental knowledge of neuronal MTs. The current proposal provides new insights and clues, through detailed study of neuronal MT behaviour and by examining animal models. Both approaches are key to understanding the pathogenesis of human brain disorders and testing possible therapies.