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 microtubule-associated 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.
Highlights
Neurons are polarized cells presenting two distinct compartments, dendrites and an axon
HMW microtubuleassociated protein-2 (MAP2) was found enriched in a subfraction containing p63 and ribophorin, two proteins mainly found in the rough endoplasmic reticulum (RER) compartment (37, 50 –53)
It seemed unlikely that MAP2 in the Rough Microsomes (RM) subfraction was associated only with mitochondria because: 1) the subfraction I3 presented an amount of VDAC similar to that of the RM subfraction, but the amount of MAP2 in this subfraction was much lower than that in the RM subfraction; and 2) electron microscopy morphometry analysis confirmed that this subfraction contained very few mitochondria
Summary
Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. 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. Despite the fact that MAP2 and tau are known to induce microtubule formation in neurons, their precise role in the elaboration of dendrites and axon remains elusive Each of these proteins presents different isoforms that are generated by alternative splicing. The presence of a distinct class of MAPs in dendrites and axon suggests that these proteins may have another function than the stabilization of microtubules Consistent with this hypothesis, microtubule formation by MAP2 and tau is not sufficient to induce process outgrowth in Sf9 cells [17, 18, 22]. One can conclude that MAP2 plays an important role in the polarized distribution of signaling proteins that regulate dendritic differentiation and plasticity
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