Major Dense Line Research Articles

The intrinsically disordered protein glue of the myelin major dense line: Linking AlphaFold2 predictions to experimental data

Numerous human proteins are classified as intrinsically disordered proteins (IDPs). Due to their physicochemical properties, high-resolution structural information about IDPs is generally lacking. On the other hand, IDPs are known to adopt local ordered structures upon interactions with e.g. other proteins or lipid membrane surfaces. While recent developments in protein structure prediction have been revolutionary, their impact on IDP research at high resolution remains limited. We took a specific example of two myelin-specific IDPs, the myelin basic protein (MBP) and the cytoplasmic domain of myelin protein zero (P0ct). Both of these IDPs are crucial for normal nervous system development and function, and while they are disordered in solution, upon membrane binding, they partially fold into helices, being embedded into the lipid membrane. We carried out AlphaFold2 predictions of both proteins and analysed the models in light of experimental data related to protein structure and molecular interactions. We observe that the predicted models have helical segments that closely correspond to the membrane-binding sites on both proteins. We furthermore analyse the fits of the models to synchrotron-based X-ray scattering and circular dichroism data from the same IDPs. The models are likely to represent the membrane-bound state of both MBP and P0ct, rather than the conformation in solution. Artificial intelligence-based models of IDPs appear to provide information on the ligand-bound state of these proteins, instead of the conformers dominating free in solution. We further discuss the implications of the predictions for mammalian nervous system myelination and their relevance to understanding disease aspects of these IDPs.

Cryo-EM, X-ray diffraction, and atomistic simulations reveal determinants for the formation of a supramolecular myelin-like proteolipid lattice

Myelin protein P2 is a peripheral membrane protein of the fatty acid-binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 β-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.

Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line

Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBP-driven formation of the MDL remains elusive at the biomolecular level. We employed complementary biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions.

Stereological quantification of age-related changes in myelinated fibers of rat white matter.

We investigated changes in myelin sheaths in the myelinated fibers of subcortical white matter in young (6-8 months old), middle-aged (18 months old), and elderly (27-28 months old) female Long-Evans rats using transmission electron microscopy and unbiased stereological techniques. We observed three age-related changes in myelin sheaths in the white matter of elderly rats: local splitting of the major dense line, myelin ballooning, and the formation of abundant myelin sheaths. Stereological analysis showed that the total length of myelinated fibers in white matter was 115±12 km (mean±SD) in young rats, 135±21 km in middle-aged rats, and 62±11 km in elderly rats. Myelinated fibers with diameters less than 1.0 μm and myelin sheath thicknesses less than 0.1 μm were significantly shorter in middle-aged and elderly rats compared with young rats. Age-related changes in myelin sheaths were evident in aged white matter. The loss of myelinated fibers with both small diameters and thin myelin sheaths is a potentially important change in aged white matter. Our results suggest that the myelin sheath changes in aged white matter may have important implications for age-related cognitive impairments.

Original article Cellular mechanisms of white matter regeneration in an adult dysmyelinated rat model

Cellular mechanisms of regeneration after the white matter injury are difficult to study because of severe, inflammatory response to massively damaged myelin. Myelin-lacking CNS of the adult Long Evans Shaker (LES) rat supplies a model where neuroregeneration can be studied conveniently. The crush site in the dorsal spinal column in LES rats implanted with the normal rat choroid plexus was studied under the light and electron microscopy at 5 time points 3-56 days post-op. While the crush injury in normal rats resulted in severe inflammation active beyond 8 weeks, the same injury in LES rats resulted in a brief inflammation that resolved before day 7 post-op. In a clear fluid-filled crush cavity, ependymal cells from the implanted choroid plexus encased multiple regenerating axons, apparently guided them across the crush cavity and participated in establishing of a zone of neuroregeneration, morphologically similar to the white matter, at the interface of the crush cavity and the surrounding tissue of the spinal cord. Axons that were not encased by implanted cells failed to cross the crush cavity and persisted as markedly swollen end bulbs filled with organelles. At 8 weeks post-op, a large proportion of axons in the zone of neuroregeneration became myelinated by Schwann cells, likely originating from dorsal nerve roots or by oligodendrocytes that formed thin sheaths with a major dense line and likely originated from the implanted choroid plexus. The LES rat can serve as a convenient model to study mechanisms of neuroregeneration including axonal regeneration in the adult CNS injury.

Abnormal brain MRI signal in 18q-syndrome not due to dysmyelination

Background18q-Syndrome is a chromosomal disorder exhibiting various symptoms arising from the central nervous system. Brain magnetic resonance imaging (MRI) of patients with this syndrome usually demonstrates abnormal white matter intensities. This is widely believed to be due to impaired myelin formation because this syndrome involves the deletion of the myelin basic protein (MBP) gene in 18q23. However, this hypothesis has not been confirmed by actual pathology because early death is unusual and autopsy rarely performed. PatientA 6-year-old boy with ring chromosome 18 syndrome was examined by genetic analysis for the MBP gene, brain MRI, and autopsy. ResultsHaploinsufficiency of the MBP gene was confirmed. T2-weighted MRI revealed diffuse high intensities throughout the cerebral white matter. Pathological examination showed the cerebral white matter to be uniformly stained by Klüver–Barrera and MBP immunohistochemical staining. Oligodendrocytes were immunoreactive for proteolipid protein and ferritin but not MBP. Electron microscopy revealed clusters of axons wrapped in compact myelin sheaths with distinct major dense lines. Holzer and immunohistochemical staining for glial fibrillary acidic protein showed extensive staining of the white matter and an increased number of glial filaments. ConclusionsThis pathological study demonstrated that in this disorder, the brain was well myelinated, contrary to established hypotheses about this disorder. The MRI signal abnormalities in 18q-syndrome could be attributed to gliosis and not to dysmyelination.

Cytoplasmic Domain of Human Myelin Protein Zero Likely Folded as β-Structure in Compact Myelin

Myelin protein zero (P0 or P0 glycoprotein), the major integral membrane protein in peripheral nervous system myelin, plays a key role in myelin membrane compaction and stability. While the structure of P0 extracellular domain was determined by crystallography, the paucity of any structural data on the highly positive-charged P0 cytoplasmic domain (P0-cyt) has greatly limited our understanding of the mechanism of P0 function. Here, using circular dichroism and intrinsic fluorescence spectroscopy, we attempted to elucidate the structure of human P0-cyt (hP0-cyt) in membrane mimetic environments composed of detergents or lipid vesicles. We found that the secondary structure of P0-cyt was polymorphic—at the lipid/protein ratio corresponding to that of mature peripheral myelin (∼50:1), hP0-cyt mainly adopted a β-conformation, whereas when the proportion of lipid increased, the structure underwent a β→ α transition. By contrast, the secondary structure of the major isoform of myelin basic protein, another myelin protein with a very large positive charge, remained unchanged across a wide range of lipid/protein ratios. We propose that when hP0-cyt is bound at sufficient concentration to lamellar lipid bilayers such as myelin, it folds into a β-conformation; before this threshold lipid/protein ratio is reached, the domain is α-helical. We suggest that the cytoplasmic apposition (major dense line) in compact myelin may be stabilized via the hydrogen-bonding of β-strands formed as a result of local P0-P0 aggregation.

Diagnostic value of ultrastructural nerve examination in Charcot-Marie-Tooth disease: two CMT 1B cases with pseudo-recessive inheritance

We report two sporadic patients of CMT disease in different consanguineous families. The electrophysiological examination led to the diagnosis of a severe demyelinating neuropathy. The nerve biopsies exhibited numerous outfoldings of the myelin sheaths and onion-bulb proliferations. The consanguinity and the histological findings pointed to a diagnosis of CMT 4B. However, the detection of abnormal and regular widenings between the major dense lines of the myelin lamellae by electron microscopy led us to search for a P0 gene mutation. Two heterozygous mutations of this gene were identified: S63F and N131Y. Different aspects of uncompacted myelin lamellae have been described in some cases of P0 mutations and a few now appear to be quite specific to it. More than 30 genes are implicated in CMT and as mutation search is time- and money-consuming, we believe that in some selected patients ultrastructural examination of nerves, among other criteria, helps orientate the molecular diagnosis of CMT.

Characterization of the murine splice variantMobp155: Developmental CNS expression pattern and subcellular localization of epitope-tagged protein

Members of the myelin-associated oligodendrocytic basic protein (MOBP) family constitute the third most abundant protein in CNS myelin. Although MOBP localizes to the major dense line (MDL) of CNS myelin, the function of the individual isoforms is unknown. Alternative splicing of pre-Mobp mRNA gives rise to six characterized splice variants in both the mouse and the rat. These splice variants share a common N-terminal encoded in Mobp exon 3 comprising 68 amino acids. The predicted protein isoforms differ in their C-termini. Sequence analysis of intron 3 revealed the presence of a putative initiation codon followed by an open reading frame (ORF) encoding 53 amino acids that extends in frame into Mobp exon 4 yielding a predicted MOBP isoform comprising 155 amino acids, designated MOBP155. This newly characterized isoform possessing a novel N-terminus shares a common C-terminus with MOBP170. Mobp170 message is detectable at low abundance throughout myelinogenesis. In contrast, the novel splice variant encoding MOBP155 is expressed at modest levels late in CNS development, coincident with the expression of the abundant splice variant, Mobp81A. Immunostaining of Cos7 cells transiently expressing an epitope-tagged MOBP155 suggested that most of the product was translocated to mitochondria. Although Mobp155 and Mobp170 encode a common predicted C-terminus they have different expression profiles and their products are targeted to mitochondria and the nucleus, respectively, in transiently transfected Cos7 cells.

Morphological changes of myelin sheaths in rats intracranially injected with apotransferrin.

Previous findings from our laboratories indicate that the intracranial injection of apotransferrin (aTf) in neonatal rats produces an accelerated oligodendrocyte maturation and an enhanced production and deposition of myelin membranes in the brain. To evaluate the anatomical distribution and the morphological characteristics of the myelin in these rats, we analyzed the optic nerves, cerebellum, and selected areas of brain sections from aTf-treated and control rats by both light and electron microscopy. Microscopic identification of myelin using a specific staining procedure, showed that in aTf-injected rats, in coincidence with previous biochemical studies, there was an increased deposition of myelin in selected areas of the nervous system. Qualitative and quantitative analysis of electron micrographs from areas showing increased myelinaton, such as the optic nerves and the corpus callosum, showed that among other changes, the intracranial treatment with aTf produces ultrastructural evidences of myelin decompaction, consisting of an enlargement in the distance between adjacent major dense lines, a decreased density of the intraperiod line, and an increased electron density of the major dense line, accompanied by a significant increase in its width. The intracranial administration of aTf induces an increased deposition of myelin by oligodeudroglial cells (OLGc), and these myelin membranes, in spite of the changes in composition and in morphology, appear to function normally. Apotransferrin can be considered as a differentiation factor that could be used to stimulate remyelination in cases in which myelin has been destroyed by various pathological processes.

Myelin P0: new knowledge and new roles.

Protein zero (P0) is an integral transmembrane glycoprotein that serves as the major protein component of peripheral nerve myelin and is a member of the immunoglobulin (IgG) gene superfamily. As a cell adhesion molecule, P0 mediates homophilic adhesive interactions between Schwann cell plasma membranes and is a key structural constituent of both the major dense line and intraperiod line of compact myelin. Both the extracellular and cytoplasmic domains contribute to these interactions and evidence indicates that the post-translational modifications of the molecule, including glycosylation, acylation and phosphorylation, play an important modulatory role in adhesion and likely in the proper trafficking of P0 from the endoplasmic reticulum to the plasma membrane as well. Structural and genetic studies indicate that mutations in P0 producing human demyelinating diseases probably do so by perturbing or preventing homophilic interactions during myelination, or by producing cellular toxicity or an unstable myelin sheath. A variety of transcription factors, growth factors and neurosteroids both directly and indirectly influence P0 gene expression during maturation of the myelinating Schwann cell. Besides its structural function in myelin, P0 may have roles in the delivery of other Schwann cell proteins to their proper location, especially at or near nodes of Ranvier, and in neuronal-glial interactions.

Myelin-associated oligodendrocytic basic protein modulates the arrangement of radial growth of the axon and the radial component of myelin

Myelin-associated oligodendrocytic basic protein (MOBP) is abundantly expressed in central nervous system myelin, and shares several characteristics with myelin basic protein (MBP), in terms of regional distribution and function. MOBP causes experimental allergic encephalomyelitis (EAE) and is associated with multiple sclerosis. MOBP has several isoforms, and the carboxy termini that are unique to each isoform contain a cluster of positively charged amino acids residues. These residues are considered to facilitate myelin sheath compaction, as does MBP. MOBP-deficient mice had compact myelin of a normal appearance. The myelin periodicity in MOBP-deficient mice was equal to that in wild-type mice. When mice were exposed to hexachlorophene, a known dysmyelinating agent, however, the myelin showed widening of the major dense lines. By morphometric analysis of the optic nerve in MOBP-deficient and MBP/MOBP-double-deficient mice, we demonstrated that MOBP played a role in controlling axonal diameter. After glutaraldehyde and tannic acid fixation, a radial series of rod-like electron enhancements in the intraperiod line in the CNS myelin, the so-called "radial component", was found arrayed in a straight radial direction in MOBP-deficient mice, while these components were scattered in an oblique zigzag direction in wild-type mice. Thus, it can be concluded that MOBP is essential for normal arrangement of the radial component.

Electron Microscopy of Myelin Basic Protein (MBP) Organized as Planar Arrays on a Lipid Monolayer Surface: Deimination of MBP Hinders Its Organizational Properties

Abstract Myelin basic protein (MBP) is one of the principal constituents of the mammalian myelin sheath. It is a basic peripheral membrane protein in the major dense line and is believed to play a structural role in maintaining myelin stability through its close association with lipids and other proteins. Immune responses to MBP have been implicated in the pathogenesis of multiple sclerosis (MS), the most common autoimmune disease of the central nervous system in North America and Northern Europe. The correlation between the severity of MS and the deimination of arginyl to citrullinyl residues in MBP was well illustrated in the acute case of MS (Marburg type) that contained MBP with 18 out of 19 arginines citrullinated. We have studied a recombinant hexahistidine-tagged murine MBP (rmMBP, 18.5 kDa isoform) by electron microscopy of the protein organized as planar arrays on lipid monolayers that consisted of the nickel chelating lipid, l,2-dioleoyl-.yn-glycero-3-[(N(5-amino-l-carboxypentyl)iminodiacetic acid)succinyl] (Nickel salt) (Ni2+-NTADOGS), and the filler lipid, liver phosphatidylinositol (PI).

Fyn tyrosine kinase participates in the compact myelin sheath formation in the central nervous system.

The cellular mechanisms for spiral wrapping and compaction of myelin sheaths by oligodendrocytes are not known yet. In this study, we examined the role of fyn tyrosine kinase, which could be responsible for molecular events during the stage of myelination in the CNS. Western blot and immunohistochemical analyses revealed that fyn-deficient mice have significantly lower levels of myelin basic protein (MBP), which is required for intracellular membrane adhesion parts so-called major dense line (MDL) and thought to be essential for the stability of myelin sheath. Electron microscopy verified that the myelin ultrastructure could be used to distinguish fyn-deficient mice from wild-type mice, showing a thin and redundant myelin sheath in the corpus callosum. Further, the electron-dense 'major' line in myelin from the purified myelin fractions remained condensed, and myelin compaction was split opened in fyn-deficient mice. To determine whether there was a change in the microheterogeneity of MBP due to a post-translational event we first investigated peptidylarginine deiminase (PAD), which is an enzyme that converts arginine residues in peptides to citrulline residues. PAD immunoreactivity was observed both in the myelin from fyn-deficient and wild-type mice. By Western blot analysis we found an increase of the citrullined form of MBP. In addition, MBP from fyn-deficient mice did weakly induce vesicle aggregation properties of MBP-mediated adhesion. We concluded that although oligodendrocytes from fyn-deficient mice are able to wrap around the axon, they are unable to form compact myelin due to decreased MBP level and the presence of increased citrullinated MBP.

Ultrastructural Localization of Monoclonal Antiphospholipid Antibody Binding to Rat Brain

Antiphospholipid antibodies (aPL), in the presence or absence of systemic lupus erythematosus, are associated with a number of neurologic complications. However, the role aPL play in pathology is unclear. A thrombotic etiology seems likely for many associated disorders, but not for others. Here we describe aPL-reactive sites in the central nervous system (CNS). Previously, using light microscopy, we showed direct binding of two monoclonal phosphatidylserine-reactive antibodies (aPS) to ependyma and myelin of fixed cat brain. In this study we determined the ultrastructural localization of their binding sites in rat CNS using immunogold electron microscopy techniques. Both monoclonal antibodies reacted strongly with myelin, preferentially with the major dense line formed by the cytoplasmic apposition of the oligodendrocyte plasma membrane. Both monoclonal antibodies also reacted with an antigen that appears associated with the axoneme in cilia of ependymal and choroid plexus epithelium. One monoclonal aPS also showed some reactivity with brain vascular endothelium and reacted slightly with mitochondria, while the other aPS did not react with these structures. While the etiology of aPL-associated neurologic disorders remains unclear, our data suggest possible target sites within the CNS with which aPL can react.

Effects of aging on myelinated nerve fibers in monkey primary visual cortex.

In monkeys, myelin sheaths of the axons in the vertical bundles of nerve fibers passing through the deeper layers of primary visual cortex show age-related alterations in their structure. These alterations have been examined by comparing the myelin sheaths in young monkeys, 5-10 years old, with those in old monkeys, between 25 and 33 years of age. The age-related alterations are of four basic types. In some sheaths, there is local splitting of the major dense line to accommodate dense cytoplasm derived from the oligodendrocytes. Other sheaths balloon out, and in these locations, the intraperiod line in that part of the sheath opens up to surround a fluid-filled space. Other alterations are the formation of redundant myelin so that a sheath is too large for the enclosed axon and the formation of double sheaths in which one layer of compact myelin is surrounded by another one. These alterations in myelin increase in frequency with the ages of the monkeys, and there is a significant correlation between the breakdown of the myelin and the impairments in cognition exhibited by individual monkeys. This correlation also holds even when the old monkeys, 25 to 33 years of age, are considered as a group. It is suggested that the correlation between the breakdown of myelin in the old monkeys and their impairments in cognition has not to do specifically with visual function but to the role of myelin in axonal conduction throughout the brain. The breakdown of myelin could impair cognition by leading to a change in the conduction rates along axons, resulting in a loss of synchrony in cortical neuronal circuits.

ALTERED DOSAGE OF PERIPHERAL MYELIN PROTEIN 22 (PMP22) AFFECTS MYELIN PERIODICITY

PMP22 is a 22‐kD glycoprotein expressed by myelinating Schwann cells. PMP22 seems to play a dual role in regulating cell growth and in peripheral myelin compaction. Myelin formation and maintenance are the result of interactions between PMP22 and the peripheral myelin protein zero (P0), which form complexes at the myelin membrane and mutually contribute to stabilize the intraperiod lines and to held together the major dense lines. Both PMP22 and P0 mutations result in specific inherited demyelinating neuropathies, which are neuropathologically characterized by uncompaction of myelin lamellae and splitting of the major dense lines in some myelinated fibers (MF). However, the most common hereditary neuropathies, Charcot‐Marie‐Tooth 1A (CMT1A) and Hereditary Neuropathy with liability to Pressure Palsies (HNPP), are due to a duplication and a deletion respectively, of the PMP22 gene, leading to over‐ and underexpression of the protein. It is unknown how the altered PMP22 dosage impairs peripheral myelination. Using our imaging system for nerve morphometry directly connected to the electron microscope, we evaluated myelin lamellae periodicity in 150 MF from sural nerves of: 3 CMT 1A patients, 3 HNPP patients, and 3 normal controls. Furthermore, we measured myelin period in 50 MF obtained from a CMT1A culture model and in 50 MF from normal DRG cultures. Myelin periodicity was significantly increased in CMT1A patients (9.77 ± 0.9 nm) and in the CMT1A culture model (10.1 ± 0.1 nm) compared respectively to the normal sural nerves (9.1 ± 0.7 nm) and to the control cultures (9.6 ± 0.1 nm). Conversely, myelin periodicity was slightly reduced in HNPP patients (8.9 ± 0.7 nm).In conclusion, PMP22 overexpression induces widening of myelin lamellae, which may be recorded with appropriate morphometric techniques. As this change has been also observed in DRG cultures from a CMT1A rat model, it may be the first stage of the demyelination process going on in this disease. The observation that altered dosage of PMP22 significantly affects myelin periodicity provides further clues in understanding the role of this protein in the process of myelin assembly and maintenance.

Multilamellar packing of myelin modeled by lipid-bound MBP.

Membrane compaction and adhesion at the major dense line (cytoplasmic apposition) of myelin, particularly in the central nervous system (CNS), is typically attributed to myelin basic protein (MBP). To explore the role of MBP in myelin membrane adhesion, we attempted to reconstitute the major dense line of myelin from purified lipid-bound MBP, which is a detergent-soluble form of MBP that retains the binding of all the myelin lipids. Removal of detergent by long-term dialysis yielded a precipitate, which, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and thin-layer chromatography, contained MBP that was still associated with myelin lipids, but in different proportions than in the native membrane. Comparison of lipid composition among isolated myelin, MBP-free myelin lipids, and lipid-bound MBP aggregates showed that the lipid-bound form of the protein was specifically enriched in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylinositol, and phosphatidylserine. Electron microscopy and x-ray diffraction demonstrated that the lipid-MBP complexes formed multilayers having periods of 70-85 A, which correspond in width to individual myelin membranes. By contrast, the lipids alone assembled as multilayers having a period of approximately 40 A. Thus, the detergent-soluble form of MBP, which is bound to lipids, might serve as a simple model for the cytoplasmic apposition of myelin.

Adhesion of acidic lipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelin basic protein

Myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces of compact myelin to give the major dense line. The 17 and 21.5 kDa isoforms containing exon II have been reported by others to localize to the cytoplasm and nucleus of murine oligodendrocytes and HeLa cells while the 14 and 18.5 kDa isoforms lacking exon II are confined to the plasma membrane. However, we show that the exon II − 18.5 kDa form and a recombinant exon II + 21.5 kDa isoform both caused similar aggregation of acidic lipid vesicles, indicating that they should have similar abilities to bind to the intracellular lipid surface of the plasma membrane and to cause adhesion of those surfaces to each other. The circular dichroism spectra of the two isoforms indicated that both had a similar secondary structure. Thus, both isoforms should be able to bind to and cause adhesion of the cytosolic surfaces of compact myelin. The fact that they do not could be due to differences in post-translational modification in vivo, trafficking through the cell and/or subcellular location of synthesis, but it is not due to differences in their lipid binding.

Messenger RNAs for kinesins and dynein are located in neural processes.

The nervous systems of all jawed animals are fully myelinated. During development, the largest caliber axons are separated from smaller neighbors by oligodendrocyte (OL) processes that envelop them with compacted multilayered myelin sheaths. Usually a single OL myelinates many axon segments. Proteins used in the sheaths are synthesized at two sites, OL soma and OL processes. Myelin basic protein (MBP) and isoforms of a second small, highly basic protein, myelin-associated oligodendrocytic basic protein (MOBP) are the only proteins known to be synthesized in OL processes. The synthesis of these two proteins is ideally situated to coordinate the compaction of the cytoplasmic leaflets into the major dense line. As a prelude to their synthesis in OL processes, the mRNAs encoding MBP and MOBP must be transported to each of the sites where myelin sheaths are formed. Morphologically, these sites are thin cytoplasmic fingers, called outer tongue processes, that overlie the compacted myelin. When one homogenizes nervous tissue, these cytoplasmic fingers become entrapped in vesicles that form from compacted myelin lamellae. The resulting myelin vesicles are readily purified by subcellular fractionation. Because they trap cytoplasm derived from OL processes, they have high levels of MBP mRNA (1). In contrast, they contain relatively little of the mRNAs that originate in other neural cell compartments, including OL soma, astrocytes, neuronal soma, and their dendritic processes (2, 3). We used mRNAs purified from a low-speed supernatant (S) of homogenized rat brain and mRNAs purified from the myelin fraction (M)-material that accumulates at a 0.25 sucrose/0.85 sucrose interface-as starting materials for suppression-subtractive hybridization (4). Briefly, the S mRNA is used to make double-stranded cDNA driver, and M mRNA is used to make M cDNA tester. Both cDNAs are digested with RsaI to make small pieces. Different adaptors are ligated to each of two separate batches of digested M cDNAs. One round of hybridization is performed in which each batch of M cDNA is melted and annealed with excess S cDNA. A second hybridization is performed with the individual hybridization reactions combined. Then, the double-stranded cDNAs, which are derived from the separate testers (i.e., they have different adaptors at each end), are selectively amplified by nested PCR. All of the above protocols follow the Clontech kit manual. The PCR product, which represents mRNAs enriched in myelin, is incorporated into vector, transformed into bacteria, and colonies that represent individual cDNAs are screened by southern blot hybridization with full-