CAMSAP1: relationship of structure to function in the process of neurite outgrowth

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences


To form a functioning nervous system, nerve cells must make connections with other cells so as to send and receive signals. They do this by extending long 'neurites' that grow away from the cell body until they find their target cell and adhere to it. The processes by which neurites grow are therefore absolutely fundamental to the formation and function of nervous systems. In our recent work funded by BBSRC, we have discovered a previously unknown protein that is an essential part of the process of neurite outgrowth. We have identified the gene that encodes this protein, and have found that blocking the expression of this gene stops the extension of neurites. In this project, we aim to learn more of the mechanism(s) that link this protein to neurite outgrowth. In particular, we wish to discover how the protein works together with other components of the process. The outcome of this project will be new insight into the mechanisms that underlie all function in nervous systems.

Technical Summary

The formation of nervous systems requires the growth from the bodies of nerve cells of long neurites (axons and dendrites) that connect with target cells. In work funded by BBSRC, we have discovered a novel protein required for neurite outgrowth from the model cell line PC12. This protein, CAMSAP1, is a calmodulin-regulated spectrin-associated protein. In this project, we aim to define aspects of the mechanism that links CAMSAP1 to the process of neurite outgrowth. In particular, we will investigate the relationship between spectrin and calmodulin binding to CAMSAP1 and the process of neurite outgrowth. The main cell model for these studies will be rat PC12 cells, but we will cross-compare results using this cell line with primary neural cultures from rats. Specifically, we aim to: 1. Define the domains of CAMSAP1 that are essential for neurite outgrowth 2. Define amino acid residues in CAMSAP1 that are essential for binding to spectrin and calmodulin by mutagenesis. 3. Compare the activities of wild-type and CAMSAP1 mutants that do not bind spectrin or calmodulin in neurite outgrowth. 4. Define further the interactions of CAMSAP1 by pull-down assays and immunoprecipitation. The outcome of this project will be novel insight into the mechanisms that link CAMSAP1, spectrin and calmodulin regulation to neurite outgrowth.


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Description The project built on a previous grant (BBSRC C18062) in which we discovered the protein CAMSAP1 (Ca2+-calmodulin-regulated microtubule and spectrin-associated protein 1). We had previously shown that CAMSAP1 is a spectrin-binding protein required for neurite outgrowth. In this project, we discovered a major microtubule-binding activity in the C-terminal region of CAMSAP1. This region contained a domain we named CKK (entered in Pfam as PF08683). The isolated CKK domain inhibited the growth of nerve axons, presumably by competing with endogenous microtubule binding proteins. Further, we identified the spectrin-binding region within a small 50 amino acid domain we named CC1 (CAMSAP-conserved 1). Within CC1, spectrin bound to 20 amino acids close to the N-terminal of CC1, and 4 amino acids within this were essential for binding. Ca2+-calmodulin also bound to CC1, within 14 residues close to the C-terminal of CC1. Spectrin and Ca2+-calmodulin competed for binding. Isolated CC1 over-expressed in neurons and PC12 cells inhibited neurite/axon outgrowth. A mutant CC1 which did not bind spectrin did not inhibit outgrowth. CC1 bound specifically to the long C-terminal isoform of beta-II spectrin in a region adjacent to the PH domain. Native EGFP-human CAMSAP1 cDNA could rescue rat CAMSAP1 siRNA knockdown of neurite outgrowth. Mutant cDNAs lacking either spectrin-binidng activity or CKK domain could not rescue neurite outgrowth. We conclude that CAMSAP1 is an essential part of the apparatus that allows nerve cells to grow axons.

We noted that the CC1 and CKK domains were common to three related human genes that we named CAMSAPs1-3. They also contain an unusual calponin homology domain (entered in Pfam as PF11971. ) Evolutionary analysis revealed that CAMSAPs (defined as CKK-CC1-CH proteins) appeared early in animal evolution, perhaps as part of the solution to organising the cytoskeleton for tissue function.

We also examined spectrin evolution, and found that spectrin first appeared in the immediate ancestors of the animals, represented in modern Choanoflagellate lineages. Spectrin appeared before its major partners (ankyrin, 4.1, adducin and CAMSAP), and seems to have been adapted for tissue function, beyond its prototypical actin cross-linking activity, during the evolution of tissues as its partners emerged, by acquiring novel protein biding sites.

We also continued an earlier collaboration on spectrin-binding proteins in heart muscle (Pinder KCL; Yacoub, Harefield). Although we found little CAMSAP1 in heart muscle, spectrin was abundant, as was protein 4.1. We characterised the 4.1 proteins of heart in detail and discovered a novel link to the Ca2+-homeostasis system via SERCA2.
Exploitation Route How do CAMSAPs fit in the processes of neurodegeneration characterised by axon death or loss?
Sectors Pharmaceuticals and Medical Biotechnology

Description We maintain an ongoing dialogue with Pfizer (now Neusentis) on our research. The findings we described to them sparked interest in both our understanding of neurite/axon growth process and the role of the cytoskeleton in membrane protein expression. A primary concern of theirs is accurate cell models for drug discovery, and we went on to collaborate with them via a BBSRC IIP in 2010 and via an industrial CASE studentship on the role of the culture environment on axon outrgowth and phenotype, taking advantage of both our expertise in aspects of cell culture, and our detailed knowledge of axonal markers and our own antibodies. They are also interested in the expression of membrane targets in recombinant cells. A further BBSRC IIP in 2011-12 deriving from our interest in spectrin and the 4.1 proteins investigated the use of cytoskeletal proteins in membrane target expression and activity.
First Year Of Impact 2009
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Description BBSRC Industrial CASE
Amount £75,281 (GBP)
Funding ID BB/I532337/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2010 
End 02/2016
Description BBSRC Industrial Interchange Programme
Amount £108,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2011 
End 06/2012
Description BBSRC Industrial Interchange Programme 2010
Amount £55,000 (GBP)
Funding ID IIP2010 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2010 
End 06/2011
Description Pfizer IIP 
Organisation Pfizer Ltd
Department Neusentis Pfizer
Country United Kingdom 
Sector Private 
PI Contribution Culture of recombinant cells expressing cytoskeletal proteins along with axonal pain targets; measurement of pain target expression
Collaborator Contribution This was partly funded by a BBSRC IIP, but the collaboration is ongoing. Neusentis provided expertise, access to proprietary cell lines and laboratory facilities.
Impact Multidisciplinary: cell culture, recombinant DNA, electrophysiology; immunological analysis.
Start Year 2011
Description Kent Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Talks and demonstrations generated much interest and questions afterwards.

Invitations to talk in schools and to local societies followed
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
Description MBP2 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact MBP2 is a project between the School of Biosciences at Kent, and Simon Langton Boys School in Canterbury, funded by the Wellcome Trust. The aim is to teach the school students some of the basic skills required to carry out front-line research into human diseases - in this specific case Multiple Sclerosis (MS). The PD RA on this project spent time at Langton participating in this project.

This stimulated great interest, especially in bioinformatic analyses.
Year(s) Of Engagement Activity 2008,2009,2010