Messenger RNAs for Kinesins and Dynein are Located in Neural ProcessesAuthor(s): Robert Gould, Concetta Freund, Frank Palmer, Pamela E. Knapp, Jeff Huang,Hilary Morrison and Douglas L. FeinsteinSource: Biological Bulletin, Vol. 197, No. 2, Centennial Issue: October, 1899-1999 (Oct., 1999),pp. 259-260Published by: Marine Biological LaboratoryStable URL: http://www.jstor.org/stable/1542638 .

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Reference: Biol. Bull. 197: 259-260. (October 1999)

Messenger RNAs for Kinesins and Dynein are Located in Neural Processes Robert Gould (NYS Institute for Basic Research, Staten Island, New York), Concetta Freund, Frank Palmer,

Pamela E. Knapp', JeffHuang2, Hilary Morrison3, and Douglas L. Feinstein4

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 sin- gle 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 trans- ported 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 pro- cesses, 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 M sucrose/0.85 M sucrose interface-as starting materials for suppression-subtrac- tive 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 per- formed 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 repre- sents 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-

1 Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky.

2 Brookdale Center for Developmental and Molecular Biology, Mount Sinai Medical Center, New York, New York.

3 Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biology Laboratory, Woods Hole, Massachusetts.

4 Department of Anesthesiology, University of Illinois, Chicago, Illinois.

length cDNA probes of the mRNAs for MBP and MOBP. The fact that 60%-80% of the cDNAs obtained from the transformed cells are derived from MBP and MOBP mRNAs is proof that subtrac- tive hybridization selectively purifies the mRNAs from OL pro- cesses.

Among the cDNAs that were sequenced were ones with signif- icant identities to KIF1A (5), a molecular motor involved in

anterograde transport of synaptic vesicles (6), and to dynein light intermediate chain protein (DLIC-2), a constituent of the dynein motor protein complex (7). Northern blot analysis studies were used to confirm that the mRNAs are concentrated in rat brain

myelin (Fig. 1). Probes to MBP and 2', 3'-cyclic nucleotide

3'-phosphodiesterase (CNP) mRNAs served as controls. MBP mRNA, located in OL processes, is enriched in rat brain myelin, whereas CNP mRNA, located in OL soma, is not (8). Here we show that mRNAs to two high molecular weight bands of KIF1A and one of two DLIC-2 mRNAs are enriched in myelin (M > S).

DNA sequences known from five other neuronal kinesin family mRNAs were used as probes in a Northern blot analysis to deter- mine whether any other kinesins were enriched in myelin. Mes-

senger RNA isoforms of two other kinesins, KHC and KIF2, were found to be enriched in myelin (data not shown). The mRNA for the major KHC was quite large, 7-9 kb, whereas that for KIF2 was small, less than 2.5 kb. Northern blots were used to determine the tissue distributions and developmental appearances of these mRNAs. The low molecular weight mRNA for KIF2 had tissue distribution and developmental expression patterns similar to those of the MBP and CNP mRNAs. Unlike these "myelination-specific" mRNAs, non-neural tissues also expressed KHC and DLIC-2 (data

MBP

S M

CNP KIF1A DLIC-2

S M S M S M

Figure 1. Equal amounts (10 ,Lg RNA/lane) of RNA from the starting material supernatant (S) and myelin fraction (M) were run on 1% agarose/ formaldehyde gels and transferred to nylon membranes. The membranes were probed with 32P-labeled cDNAsfor MBP, CNP, and two novel cDNAs obtained by subtractive hybridization (see text) according to methods used in our lab (8). We have not checked these membranes for equal loading, but have found the same results for each probe on several occasions. The sizes of the mRNAs are roughly 2 kb (MBP), 5 kb (CNP), 7 and 9 kbfor the

larger KIFIA bands and more than 6 kb for the DLIC-2 band enriched in

myelin. The sizes for MBP, CNP and DLIC mRNAs are the same as

expected from the sizes of the cDNAs (accessed through GenBank).

259 CELL MOTILITY

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REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Figure 2. In situ hybridization of KIFIA mRNA in (A) the dorsal column of PO1 rat spinal cord and in (B) cultured mouse OL. In both

pictures the arrows point to labeled cell processes. The resolution in cultured cells is sufficient to see that the mRNA is present in discrete

granules, which would be an indicator that the mRNA was transported in

granules.

Figure 2. In situ hybridization of KIFIA mRNA in (A) the dorsal column of PO1 rat spinal cord and in (B) cultured mouse OL. In both

pictures the arrows point to labeled cell processes. The resolution in cultured cells is sufficient to see that the mRNA is present in discrete

granules, which would be an indicator that the mRNA was transported in

granules.

not shown). Their developmental expression patterns also differed from MBP and CNP mRNAs; they were expressed throughout postnatal development. In situ hybridization studies confirm the presence of these mRNAs in OL processes in vivo and in culture. We have demonstrated that the KIF1A probe recognizes mRNAs in a cluster of OLs in the dorsal column of a young rat spinal cord and in cultured mouse brain OLs (Fig. 2). In cells in vivo and in culture, mRNA is clearly seen in long cell processes, indicative of mRNA transport. Synthesis of motor proteins in OL processes indicates that complex "microtubule-based" communication sys- tems are in place to transport vesicles from sites of myelin sheath assembly back to the OL soma. This system could function to

not shown). Their developmental expression patterns also differed from MBP and CNP mRNAs; they were expressed throughout postnatal development. In situ hybridization studies confirm the presence of these mRNAs in OL processes in vivo and in culture. We have demonstrated that the KIF1A probe recognizes mRNAs in a cluster of OLs in the dorsal column of a young rat spinal cord and in cultured mouse brain OLs (Fig. 2). In cells in vivo and in culture, mRNA is clearly seen in long cell processes, indicative of mRNA transport. Synthesis of motor proteins in OL processes indicates that complex "microtubule-based" communication sys- tems are in place to transport vesicles from sites of myelin sheath assembly back to the OL soma. This system could function to

transport those proteins that must be removed from the OL plasma membrane so that the myelin sheaths will be left with their select and rather simple protein composition. We hypothesize that the

appearance of KIF1A, KHC, and DLIC-2 mRNAs early in devel-

opment indicates that these proteins are formed in OL processes at

early developmental stages, i.e., when OLs first contact the axons

they myelinate. If this is the case, the motors may play a role in

transporting axon-derived material back to the OL soma.

Supported by a grant (RG2944AG/1) from the National Multi-

ple Sclerosis Society.

Literature Cited

1. Colman, D. R., G. Kreibich, A. B. Frey, and D. D. Sabatini. 1982. J. Cell Biol. 95: 598-608.

2. Gillespie, C. S., L. Bernier, P. J. Brophy, and D. R. Colman. 1990. J. Neurochem. 54: 656-661.

3. Gould, R. M. 1998. J. Neurochem. 70 Suppl. 1 S53. 4. Diatchenko, L., Y.-F. C. Lau, A. P. Campbell, A. Chenchik, F.

Mooadam, B. Huang, S. Lukyanov, K. Lukyanov, N. Gurskaya, E. D. Sverdlov, et al. 1996. Proc. Natl. Acad. Sci. USA 93: 6025- 6030.

5. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Nucleic Acids Res. 25: 3389- 3402.

6. Okada, Y., Y. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell 81: 769-780.

7. Hughes, S. M., K. T. Vaughan, J. S. Herskovits, and R. B. Vallee. 1995. J. Cell Sci. 108: 24.

8. Gould, R. M., C. M. Freund, and E. Barbarese. J. Neurochem. 73: (in press).

transport those proteins that must be removed from the OL plasma membrane so that the myelin sheaths will be left with their select and rather simple protein composition. We hypothesize that the

appearance of KIF1A, KHC, and DLIC-2 mRNAs early in devel-

opment indicates that these proteins are formed in OL processes at

early developmental stages, i.e., when OLs first contact the axons

they myelinate. If this is the case, the motors may play a role in

transporting axon-derived material back to the OL soma.

Supported by a grant (RG2944AG/1) from the National Multi-

ple Sclerosis Society.

Literature Cited

1. Colman, D. R., G. Kreibich, A. B. Frey, and D. D. Sabatini. 1982. J. Cell Biol. 95: 598-608.

2. Gillespie, C. S., L. Bernier, P. J. Brophy, and D. R. Colman. 1990. J. Neurochem. 54: 656-661.

3. Gould, R. M. 1998. J. Neurochem. 70 Suppl. 1 S53. 4. Diatchenko, L., Y.-F. C. Lau, A. P. Campbell, A. Chenchik, F.

Mooadam, B. Huang, S. Lukyanov, K. Lukyanov, N. Gurskaya, E. D. Sverdlov, et al. 1996. Proc. Natl. Acad. Sci. USA 93: 6025- 6030.

5. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Nucleic Acids Res. 25: 3389- 3402.

6. Okada, Y., Y. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell 81: 769-780.

7. Hughes, S. M., K. T. Vaughan, J. S. Herskovits, and R. B. Vallee. 1995. J. Cell Sci. 108: 24.

8. Gould, R. M., C. M. Freund, and E. Barbarese. J. Neurochem. 73: (in press).

Reference: Biol. Bull. 197: 260-262. (October 1999)

Migration Forces in Dictyostelium Measured by Centrifuge DIC Microscopy Yoshio Fukui', Taro Q. P. Uyeda2, Chikako Kitayama2, and Shinya Inoue

(Marine Biological Laboratory, Woods Hole, Massachusetts 02543-1015)

Reference: Biol. Bull. 197: 260-262. (October 1999)

Migration Forces in Dictyostelium Measured by Centrifuge DIC Microscopy Yoshio Fukui', Taro Q. P. Uyeda2, Chikako Kitayama2, and Shinya Inoue

(Marine Biological Laboratory, Woods Hole, Massachusetts 02543-1015)

Amoeboid locomotion represents an important biological activ- ity involved in cell growth and development (1). Forces that underlie movement of the giant amoeba, Chaos chaos, have been estimated to be 1.5 X 102 pN//Im2 as measured by Kamiya's double chamber method (2). For a slime mold, Dictyostelium discoideum, the forces of cell locomotion have been unknown, but the cortex resists poking with a microneedle (cortical tension) at 1.4 x 103 pN//im2 (3). By micropipette aspiration, the cortical tension of D. discoideum has been measured as 1.55 X 103 pN/jtm2 (4). In the present study, we determined the migration stalling forces of D. discoideum by using a centrifuge polarizing

1 Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3008.

2 Biomolecular Research Group, National Institute for Advanced Inter- disciplinary Research, Tsukuba, Ibaraki 305-8562, Japan.

Amoeboid locomotion represents an important biological activ- ity involved in cell growth and development (1). Forces that underlie movement of the giant amoeba, Chaos chaos, have been estimated to be 1.5 X 102 pN//Im2 as measured by Kamiya's double chamber method (2). For a slime mold, Dictyostelium discoideum, the forces of cell locomotion have been unknown, but the cortex resists poking with a microneedle (cortical tension) at 1.4 x 103 pN//im2 (3). By micropipette aspiration, the cortical tension of D. discoideum has been measured as 1.55 X 103 pN/jtm2 (4). In the present study, we determined the migration stalling forces of D. discoideum by using a centrifuge polarizing

1 Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3008.

2 Biomolecular Research Group, National Institute for Advanced Inter- disciplinary Research, Tsukuba, Ibaraki 305-8562, Japan.

microscope (CPM) equipped with DIC optics (5). The results demonstrated that individual wild type (NC4) amoebae (6) can crawl centripetally on a glass surface, resisting gravitational forces

larger than 11,465 x g. NC4 amoebae can also undergo normal

cytokinesis at forces of at least 8376 X g. Dictyostelium cells were washed with Bonner's saline solution

(BSS: 10 mM NaCl, 10 mM KC1, 3 mM CaCl3) and allowed to attach to an ethanol-cleaned glass slide in a custom centrifuge chamber filled with BSS. Frozen images of the spinning micro-

scopic field containing 20-50 cells were recorded onto Sony ED-Beta tape through an Olympus SLC Plan Fl 40x (N.A. 0.55) or LC Plan Fl 20X (N.A. 0.40) objective lens and a condenser lens (LC Plan Fl 20X/N.A. 0.40). The images illuminated by a 532-nm

pulsed laser were captured in real time with a Hamamatsu C5946 CCD camera equipped with an interference-fringe-free filter. The centrifuge disk rotates horizontally, and its speed was controlled in

microscope (CPM) equipped with DIC optics (5). The results demonstrated that individual wild type (NC4) amoebae (6) can crawl centripetally on a glass surface, resisting gravitational forces

larger than 11,465 x g. NC4 amoebae can also undergo normal

cytokinesis at forces of at least 8376 X g. Dictyostelium cells were washed with Bonner's saline solution

(BSS: 10 mM NaCl, 10 mM KC1, 3 mM CaCl3) and allowed to attach to an ethanol-cleaned glass slide in a custom centrifuge chamber filled with BSS. Frozen images of the spinning micro-

scopic field containing 20-50 cells were recorded onto Sony ED-Beta tape through an Olympus SLC Plan Fl 40x (N.A. 0.55) or LC Plan Fl 20X (N.A. 0.40) objective lens and a condenser lens (LC Plan Fl 20X/N.A. 0.40). The images illuminated by a 532-nm

pulsed laser were captured in real time with a Hamamatsu C5946 CCD camera equipped with an interference-fringe-free filter. The centrifuge disk rotates horizontally, and its speed was controlled in

260 260

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