Interaction of Microtubule-associated Protein-2 and p63

Abstract

Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubuleassociated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron.Neurons are polarized cells that present two distinct compartments, dendrites and an axon. These compartments can be distinguished by their morphology; dendrites are multiple and taper, whereas the axon is unique and its diameter is uniform (1-4). Dendrites and axon also differ by their membranous organelle composition; RER 1 is found in the somato-dendritic compartment but not in the axon, and the number of free ribosomes is a lot higher in dendrites than in the axon (1, 2, 5). The cytoskeletal elements are also distinctly distributed in these neuronal compartments; the number of microtubules is higher in dendrites than in the axon, whereas the opposite is noted for neurofilaments (1, 2, 6).Microtubules are involved in the establishment of cell polarity. Notably, the microtubules of dendrites and axon contain different microtubule-associated proteins (MAPs); the microtubule-associated protein-2 (MAP2) is found in dendrites, whereas tau is present only in the axon (6 -8). The contribution of these MAPs to the establishment of neuronal polarity has been well documented. Tau contributes to the axonal differentiation in primary neuronal cultures, whereas MAP2 is involved in the differentiation of minor neurites, the neuronal processes that become dendrites, and in the maintenance of dendrites in adult neurons (9 -14). Despite the fact that MAP2 and tau are...

Dynamic and reversible restructuring of the ER induced by PDMP in cultured cells

“…The SER random tubular network is characterised by highly convoluted tubules rather than by the relatively straight ones of the polygonal meshwork and could thus represent a spontaneous arrangement of the ER -similar to the so-called 'sponge phase' of curvature theoreticians (Hyde et al, 1997) -that is attained in the absence of regulatory factors mediating interaction with microtubules. Consistently, CLIMP-63, a membrane protein that mediates static interaction of the RER with microtubules (Farah et al, 2005;Klopfenstein et al, 1998;Schweizer et al, 1995) was excluded from the interior of the SER patches. On the basis of the considerations above, we propose a model for the PDMP-induced formation of SER patches, illustrated in Fig.…”

HMWMAP2: New perspectives on a pathway to dendritic identity

“…The function of HMWMAP2 on the surface of RER membranes is most likely to anchor these membranes to microtubules and thereby to contribute to their segregation in the somato-dendritic compartment [Bartlett and Banker, 1984a,b;Peters et al, 1991]. We showed that the interaction of HMWMAP2 with CLIMP-63 is necessary for the association of RER membranes with microtubules in an in vitro assay [Farah et al, 2005]. Several studies reported that the ER membranes are associated with microtubules [Terasaki et al, 1986;Terasaki, 1990;Terasaki and Reese, 1994;Hirokawa, 1998;Aihara et al, 2001].…”

“…The mechanisms underlying the segregation and/or the maintenance of the RER membranes in this neuronal compartment seem to involve HMWMAP2. In a recent study, we reported that HMWMAP2 are associated with the rough endoplasmic reticulum (RER) [Farah et al, 2005] and that this association is mediated by the interaction of the HMWMAP2 projection domain with the RER protein termed cytoskeleton-linking membrane protein-63 (CLIMP-63). CLIMP-63 is an integral membrane protein that links RER membranes to microtubules in non-neuronal cells [Klopfenstein et al, 1998].…”

NMDA Receptor Activation Suppresses Microtubule Growth and Spine Entry

“…The following mammalian expression plasmids have been described: pGW1-EB3-GFP (Jaworski et al, 2009), pGW1-GFP, p␤actin-HA-␤-galactosidase (Hoogenraad et al, 2005), pSuper vector (Brummelkamp et al, 2002), pSuper-EB3-shRNA (Jaworski et al, 2009), GFP-MAP2c (Farah et al, 2005), NR2B-shRNA and NR2A-shRNAs (Kim et al, 2005), mCherry-␣-tubulin (Shaner et al, 2004), and GST-EB3 (Komarova et al, 2005). GFP-KIFC2 was generated by PCR cloning full-length rat KIFC2 cDNA into pEGFP-C1.…”