Messenger RNAs for Kinesins and Dynein are Located in Neural Processes

Abstract

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 supematant (S) of homogenized rat brain and mRNAs purified from the myelin fraction (M)-material that accumulates at a 0.25 M sucrose/O.85 M 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

Messenger RNAs Located in Myelin Sheath Assembly Sites

“…The present study (see also Gould et al., 1999 b ) identifies mRNAs to several microtubule‐based motor proteins in MSAS, two with high sequence homology to KIF1A (Okada et al, 1995) and one with high sequence homology to DLIC‐2 (Hughes et al, 1995). The KIF1A and DLIC‐2 mRNAs enriched in myelin are clearly larger than the known KIF1A and DLIC mRNAs (Table 3).…”

“…Characterization of more 5′ sequences will enable us to find out if the encoded protein is larger than the known DLIC‐2. In addition to these mRNAs, we also identified mRNAs for a novel KIF2 and KIF5B that are enriched in myelin (Gould et al, 1999 b ). Thus, a variety of motor proteins are likely to be synthesized in MSAS.…”

“…Thus, a variety of motor proteins are likely to be synthesized in MSAS. In the recent study (Gould et al, 1999 b ), we used in situ hybridization to show that KIF1A mRNA is located in oligodendrocyte processes in vivo and in granules similar to those that transport MBP mRNA in cultured oligodendrocytes (Ainger et al, 1997). Further studies are needed to better define all the kinesin and dynein/dynactin family members synthesized in MSAS and to identify the organelles and destinations transported by each.…”

KIF22 promotes bladder cancer progression by activating the expression of CDCA3

“…Kinesin family members (KIFs) are a group of molecular motor proteins involved in the transport of cargo along the microtubule in an adenosine triphosphate (ATP)-dependent manner (11). KIFs mediate a variety of cellular functions, such as mitosis, ciliary assembly and signaling transduction (12)(13)(14). KIF22 is a kinesin-like DNA binding protein (15,16).…”

Neural Stem Cell Purification and Clonal Analysis

“…NSCs have also been isolated from adult regions using this method. Neurogenesis continues throughout life in a variety of species including birds, rodents, nonhuman primates and humans (Goldman and Nottebohm, 1983;Lois and AlvarezBuylla, 1993;Kempermann et al, 1997;Eriksson et al, 1998;Gould et al, 1999;Kornack and Rakic, 1999). In contrast to the widespread neurogenesis of the embryonic brain, neurogenesis in the adult brain occurs in very restricted places, the subventricular zone (SVZ) surrounding the lateral ventricles which continually feeds new neurons into the olfactory bulbs through the rostral migratory stream (RMS) and the dentate gyrus (DG) of the hippocampus (reviewed by Kempermann et al, 1997;Alvarez-Buylla and Garcia-Verdugo, 2002).…”