The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode–axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.
During myelin formation, membrane-associated proteins have to be sorted and transported in specified membrane regions such as compact and non-compact myelin membranes. One protein that may be involved in such a process is the Myelin and Lymphocyte protein MAL (VIP17/ MVP17). MAL was identified as a novel myelin membrane component expressed by oligodendrocytes and Schwann cells. Since MAL has been shown to be important in the apical sorting machinery of polarized cells, we have started to investigate the possible functional role of MAL in sorting myelin membrane-associated molecules. In this study, we have generated cDNA constructs with green fluorescent protein (GFP) either at the N- or C-terminus of MAL. Transfection experiments showed that GFP-MAL expression resembles that of normal MAL, whereas the MAL-GFP fusion construct was not properly transported within the cell. Furthermore, we could demonstrate that GFP-MAL is enriched in detergent insoluble glycolipid-enriched microdomains as already seen for untagged MAL. As a prerequisite for the generation of transgenic mice expressing GFP-MAL under the control of its own regulatory elements, we have generated a cDNA construct with an 8-kb MAL promotor fragment fused to GFP-MAL. Transfection experiments of the Oli-neu oligodendrocyte cell line showed that GFP-MAL was expressed, but only in cells, which were stimulated for differentiation with cAMP. In summary, the results confirm that the fusion protein GFP-MAL is incorporated into detergent-insoluble complexes and the 8-kb MAL promotor fragment is sufficient to be activated in oligodendrocytes.
beta-Thymosins are polypeptides that bind monomeric actin and thereby function as actin buffers in many cells. We show that during zebrafish development, β-thymosin expression is tightly correlated with neuronal growth and differentiation. It is transiently expressed in a subset of axon-extending neurons, essentially primary neurons that extend long axons, glia and muscle. Non-neuronal expression in the brain is restricted to a subset of glia surrounding newly forming axonal tracts. Skeletal muscle cells in somites, jaw and fin express beta-thymosin during differentiation, coinciding with the time of innervation. Injection of beta-thymosin antisense RNA into zebrafish embryos results in brain defects and impairment of the development of beta-thymosin-associated axon tracts. Furthermore, irregularities in somite formation can be seen in a subset of embryos. Compared to wild-type, antisense-injected embryos show slightly weaker and more diffuse engrailed staining at the midbrain-hindbrain boundary and a strong reduction of Isl-1 labeling in Rohon Beard and trigeminal neurons. The decreased expression is not based on a loss of neurons indicating that beta-thymosin may be involved in the maintenance of the expression of molecules necessary for neuronal differentiation. Taken together, our results strongly indicate that beta-thymosin is an important regulator of development.
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