FRIEDRICH PROPST, Ph.D.
phone     +43-1-4277 52858
Fax         +43-1-4277 52854
E-mail:    friedrich.propst@univie.ac.at

 

Molecular and functional analysis of microtubule-associated
proteins during differentiation in cell culture and knockout mice

During development of the nervous system, a major task for extending neurites is to recognize and heed environmental cues to follow the appropriate paths to their prospective targets. These environmental cues are read by growth cones at the tips of neurites and are interpreted to bring about the necessary changes in direction of growth cone migration. As with all forms of cell migration, growth cone migration involves constant rearrangements of the cytoskeleton, mainly microtubules and actin filaments. Thus, growth cone guidance can be viewed as the task to appropriately rearrange the microtubule and microfilament networks in response to extracellular signals. The translation of extracellular signals into rearrangements of actin filaments is well understood [13]. Much less is known about how extracellular signals lead to the essential changes in microtubule organization to ensure an appropriate growth cone response. One hypothesis is that brain-specific microtubule-associated proteins (MAPs) which were discovered more than 20 years ago might be involved in signal transduction [15,24,20,5].

Among the classical MAPs, MAP1B is a prime candidate for a signal transduction molecule involved in growth cone steering. It is predominantly expressed during brain development and localized and differentially phosphorylated in growth cones [1,3]. Candidate kinases are cdk5 and GSK-3b [7,12], both of which are implicated in signal transduction and growth cone steering. MAP1B is differentially phosphorylated depending on the presence of laminin in the extracellular matrix [19] and destruction of MAP1B by chromophore-assisted laser inactivation in subdomains of growth cones alters their response to a laminin cue [14]. Ablation of MAP1B expression by antisense oligonucleotides prevents the growth of axons in in vitro cultures of neuronal cells [2,4]. Moreover, a MAP1B-related protein in Drosophila, termed Futsch, is essential for extension of axons and dendrites in the fly [8]

The aim of our work is the molecular analysis of MAPs such as MAP1B as well as the identification of novel MAP1B related proteins involved in morphogenesis. Our approach to determine the function and mechanism of action of MAP1B is based on molecular and cellular biology, in vitro assays and the generation of transgenic and knockout mice. Functional analysis is carried out by expressing the full length proteins, deletion mutants and subdomains in a variety of mammalian tissue culture cells. The aim is to identify important functional domains and biologically relevant binding partners (complemented by yeast 2-hybrid screen and in vitro binding assays). In our experiments involving transgenic and knockout mice we are studying the effects of MAP1B deficiency in axonal guidance. Our results demonstrate that MAP1B is required for axonal guidance in certain areas of the developing brain and support the concept of MAP1B as a signal transduction molecule translating extracellular guidance cues into rearrangements of the axonal microtubule and actin filament network. Our future focus will be the analysis of axonal guidance in MAP1B deficient neurons derived from our MAP1B knockout mice in vitro by digital time-lapse microscopy and the identification and functional analysis of proteins structurally or functionally related to MAP1B by biochemical and cell biological methods and by gene targeting.

The MAP1B knockout

To determine whether MAP1B is necessary for mouse brain development we decided to re-examine controversial results obtained in previous knockout studies. We generated MAP1B knockout mice utilizing a new, unequivocal and comprehensive knockout strategy, thus avoiding the caveats of previous studies. To this end we cloned the MAP1B gene of the mouse and analyzed its expression in various tissues and at different developmental stages. We found that in addition to regular MAP1B transcripts, alternative transcripts can be detected in all tissues where MAP1B is expressed [9]. Moreover, these alternative transcripts are homologous to those found in rat and human. We concluded that alternative transcription of the MAP1B locus is conserved in evolution to give rise to N-terminally truncated MAP1B isoforms. These results explained why in one of the previous MAP1B knockout studies the synthesis of MAP1B was down regulated but not abolished and hence did not reveal the phenotype of true MAP1B deficiency. We also determined the previously unknown 3' end of MAP1B mRNA [17]. We found an unusually long and highly conserved 3'UTR of 4.3 kb and found that the MAP1B 3'UTR overlapped with the 5' end of the cDNA encoding another gene called DBI-1. However, our further analysis showed that the published structure of DBI-1 cDNA is most likely the result of fortuitous joining of unrelated cDNA fragments during cloning.

Based on these results we were able to design a new knockout strategy which removed 93% of the MAP1B coding region (Fig. 1). This resulted in a true null allele with complete ablation of MAP1B protein expression and allowed us for the first time to assess the effect of MAP1B deficiency in vivo [16]. The most striking developmental defect in our mice was the absence of the corpus callosum, a prominent commissure connecting the hemispheres of the cerebrum, and the formation of misguided axon bundles, demonstrating that MAP1B is required for axon guidance (Fig. 2). Similar defects have been observed in mice deficient for the chemotactic factor netrin-1 [21], the netrin receptor Dcc [6] and the actin regulatory protein Mena [11] as well as in mice deficient for the protein-tyrosine kinase receptor Sek4 [18] and the cdk5 kinase regulatory protein p35 [10]. This raised the possibility that MAP1B is a component of signal transduction pathways regulating microtubule-actin interaction in response to extracellular guidance cues such as netrin and laminin and their corresponding downstream signal transduction molecules (Fig. 3).

MAP1B as a signal transduction molecule

To identify and characterize possible functional domains of MAP1B we transiently transfected non-neuronal NIH3T3 fibroblasts and PtK2 cells with constructs encoding epitope-tagged full length and deletion mutant MAP1B and analyzed expressing cells by immunofluorescence, immunoblotting and immunoprecipitation. Non-neuronal cells were chosen, because they express little or no endogenous MAP1B, permitting a straight forward assessment of the effects of the ectopic wild-type or mutant MAP1B on cytoskeletal components.

The high molecular mass MAPs MAP1B and MAP1A are synthesized as polyprotein precursors which are rapidly cleaved to give rise to heavy and light chains constituting the respective MAP1B or MAP1A complex. We found that a novel hydrophilic, proline-rich 45-amino acid domain containing the cleavage site is necessary and sufficient for proteolytic cleavage of the MAP1B precursor protein [22]. This domain is conserved in MAP1A. Additional sequences in the N-terminal half of the heavy chain contribute to the efficiency of cleavage (Fig. 4).

Previous studies on the role of MAP1B in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and revealed moderate effects on microtubule stability. In contrast, we analyzed the effects of the MAP1B light and heavy chains separately and were able to assign several crucial activities to the MAP1B light chain [23]. We found that distinct from all other MAPs, the MAP1B light chain induced formation of stable but apparently flexible microtubules arranged in bundles and loops, resembling microtubule networks found in neuronal growth cones (Fig. 5). The MAP1B light chain-modified microtubules were found to be stabilized against the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its carboxy terminus. By coimmunoprecipitation experiments we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain-light chain interaction to regions containing sequences homologous to MAP1A. Our findings (summarized in Fig. 6) suggested a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity (Fig. 7).
 

References

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[16]    Meixner, A., Haverkamp, S., Wässle, H., Führer, S., Thalhammer, J., Kropf, N., Bittner, R.E., Lassmann, H., Wiche, G. and Propst, F. MAP1B is required for axon guidance and is involved in the development of the central and peripheral nervous system (2000) J. Cell Biol. 151, 1169-1178.

[17]    Meixner, A., Wiche, G. and Propst, F. Analysis of the mouse MAP1B gene identifies a highly conserved 4.3 kb 3' untranslated region and provides evidence against the proposed structure of DBI-1 cDNA (1999) Biochim. Biophys. Acta 1445, 345-350.

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[22]    Tögel, M., Eichinger, R., Wiche, G. and Propst, F. A 45-amino acid residue domain necessary and sufficient for proteolytic cleavage of the MAP1B polyprotein precursor (1999) FEBS Lett. 451, 15-18.

[23]    Tögel, M., Wiche, G. and Propst, F. Novel features of the light chain of microtubule-associated protein MAP1B: microtubule stabilization, self interaction, actin filament binding, and regulation by the heavy chain (1998) J. Cell Biol. 143, 695-707.

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Department of Molecular Cell Biology