Centennial Issue: October, 1899-1999 || Migration Forces in Dictyostelium Measured by Centrifuge DIC Microscopy

Migration Forces in Dictyostelium Measured by Centrifuge DIC MicroscopyAuthor(s): Yoshio Fukui, Taro Q. P. Uyeda, Chikako Kitayama and Shinya InouéSource: Biological Bulletin, Vol. 197, No. 2, Centennial Issue: October, 1899-1999 (Oct., 1999),pp. 260-262Published by: Marine Biological LaboratoryStable URL: http://www.jstor.org/stable/1542639 .

Accessed: 28/06/2014 07:33

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Marine Biological Laboratory is collaborating with JSTOR to digitize, preserve and extend access toBiological Bulletin.

http://www.jstor.org

This content downloaded from 46.243.173.188 on Sat, 28 Jun 2014 07:33:37 AMAll use subject to JSTOR Terms and Conditions

http://www.jstor.org/action/showPublisher?publisherCode=mbl

http://www.jstor.org/stable/1542639?origin=JSTOR-pdf

http://www.jstor.org/page/info/about/policies/terms.jsp


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 systems 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 systems 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)


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



CELL MOTILITY

Table I

Migration stall forces in Dictyostelium

Strain*

Measurement NC4 Ax3 HS1 A5

A. Reduced mass of different strains and myosin mutants Radius (X10-4 cm) 4.81 + 0.95 5.47 + 1.05 5.14 + 0.83 5.48 ? 0.98 Volume (X10-10 cm3) 3.78 ? 1.44 4.87 + 1.80 4.26 ? 1.34 4.87 + 1.69 AMasst (X10-11 g) 2.57 ? 0.98 3.32 + 1.23 2.90 ? 0.91 3.31 ? 1.15

B. Migration stall forces of different strains and myosin mutants

Maximum Rotation (rpm)t >11,700 6,400 3,500 3,400 Gravity (X g) >11,465 3,431 1,025 968 Force ? (x103 pN) >8.77 ? 1.10 1.08 + 0.42 0.28 ? 0.09 0.30 + 0.11

C. Migration stall forces and medium density in HS1

Percoll (%) 0% 10% 25% 50% 75% 100% Density (g/ml) 1.000 1.024 1.032 1.064 1.104 1.186 Maximum Rotation (rpm)t 3,500 4,100 4,900 6,800 7,800 8,600 Gravity (X g) 1,025 1,408 2,011 3,873 5,096 6,195

* NC4: wild type, Ax3: axenic mutant, HS1: myosin II knock-out mutant, A5: triple (myoIA, myoIB, myosin II) knock-out mutant. t AMass = (Cell density - Medium density) X Volume = (1.068 - 1.000) X Volume = 0.068 (g/cm3) X Volume (cm3) $ Maximum rotor rpm beyond which the amoebae were unable to crawl centripetally. ? Standard deviation each based on measurements of diameters of more than 100 cells.

100-rpm increments up to a maximum speed of 11,700 rpm. The radius from the center of the disk to the center of the observation chamber was 7.5 cm. We determined the maximum rotation speed at which the cell's geometric center ("centroid") exhibited centripetal movement, i.e., movement towards the center of the rotor. The measurement was done for wild type (NC4) (6), axenic strain (Ax3) (6), and two myosin knock-out mutants. Of the two, HS1 is a myosin II null mutant that does not express conventional myosin, which is responsible for production of major mechanochemical forces (7). A5 is a triple knock-out mutant that does not express myoIA, myoIB, or myosin II (8).

Migration forces were calculated from Newton's Force Law, i.e., F = m X a, where F is force (in pico Newton: pN), m is mass (in grams), and a is acceleration (in centimeters per second squared). We measured cell volumes from the diameter of round cells and calculated the reduced mass by multiplying the volume by the density difference (1.068 - 1.000 g/cm3) (9). The average radius, calibrated volume, and reduced mass of NC4, Ax3, HS1, and A5 are shown in Table IA. As shown, the maximum rotational speeds at which the amoebae were able to crawl centripetally were 11,700, 6400, 3500, and 3400 rpm, respectively. No centripetal migration occurred when the rotor speed was increased by 100

rpm. These values correspond to 11,465 X g, 3431 X g, 1025 X

g, and 968 X g, respectively. These results showed that the gravitational forces equivalent to the migration stall forces are, respectively, >2.77 X 103 pN, 1.08 X 103 pN, 0.28 X 103 pN, and 0.30 X 103 pN (Table IB).

We also examined the "maximum rotation speed" as a function of density of the medium (Table IC). The results of these experi- ments were unexpected; the ability of the amoebae to migrate centripetally continued to increase with the density of the medium,

even when it substantially exceeded the density of the amoebae themselves, so that detached amoebae would float. Since all strains exhibited the same level of adhesion up to maximum rotation (i.e., 11,700 rpm), we propose that the capacity for centripetal movement in fact represents the migration forces of those amoebae. The behavior of an amoeba in a medium with a density greater than its own must signify a stalling mechanism based not on the overall buoyant density of the amoeba, but perhaps on some stratified components on or within the amoeba.

This study demonstrates that the axenic strain (Ax3) is in fact a

type of mutant (10) that can generate less than 39% of the migration force generated by the original wild type (NC4). This study further demonstrates that a myosin II knock-out mutant (HS1) can generate only 26% of the migration force that its axenic parent (Ax3) can produce. In contrast, knocking-out myoIA and IB (A5) produces no additional decrease in the generation of migration forces. The migration stall forces exhibited by those mutants are obviously not dependent on myoIA, myoIB or myosin II, suggest- ing a significant contribution by other actin-based, force-generat- ing mechanisms.

Literature Cited

1. Fukui, Y. 1993. Int. Rev. Cytol. 144: 85-127. 2. Kamiya, N. 1964. Pp. 257-277 in Primitive Motile Systems in Cell

Biology, R. D. Allen and N. Kamiya, eds. Academic Press, New York. 3. Pasternak, C., J. A. Spudich, and E. L. Elson. 1989. Nature 341:

549-551. 4. Gerald, N., J. Dai, H. P. Ting-Beall, and A. De Lozanne. 1998.

J. Cell Biol. 141: 483-492. 5. Goda, M., S. Inou6, and R. Knudson. 1998. Biol. Bull. 195:

212-214.

261



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

6. Raper, K. B. 1984. Pp. 19, 74-75 in The Dictyosteliads, Princeton University Press. Princeton, New Jersey.

7. Ruppel, K. M., T. Q. P. Uyeda, and J. A. Spudich. 1994. J. Biol. Chem. 269: 18773-18780.

6. Raper, K. B. 1984. Pp. 19, 74-75 in The Dictyosteliads, Princeton University Press. Princeton, New Jersey.

7. Ruppel, K. M., T. Q. P. Uyeda, and J. A. Spudich. 1994. J. Biol. Chem. 269: 18773-18780.

8. Kitayama, C., J. Dai, H. P. Ting-Beall, M. A. Titus, and T. Q. P. Uyeda. 1998. Mol. Biol. Cell 9: 387a.

9. Fukui, Y. 1976. Dev. Growth Differ. 18: 145-155. 10. Kayman, S. C., and M. Clarke. 1983. J. Cell Biol. 97: 1001-1010.

8. Kitayama, C., J. Dai, H. P. Ting-Beall, M. A. Titus, and T. Q. P. Uyeda. 1998. Mol. Biol. Cell 9: 387a.

9. Fukui, Y. 1976. Dev. Growth Differ. 18: 145-155. 10. Kayman, S. C., and M. Clarke. 1983. J. Cell Biol. 97: 1001-1010.


Dynamic Confocal Imaging of Interphase and Mitotic Microtubules in the Fission Yeast, S. pombe P. T. Tran', P. Maddox2, F. Chang', and S. Inoue

(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)


Dynamic Confocal Imaging of Interphase and Mitotic Microtubules in the Fission Yeast, S. pombe P. T. Tran', P. Maddox2, F. Chang', and S. Inoue

(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

In the fission yeast, S. pombe, microtubules are required for multiple cellular processes, including maintenance of cell polarity, positioning of cellular organelles, and mitosis. Thus, microtubules are dynamic polymers (1), remodeling themselves within the living cell throughout the cell cycle.

Unfortunately, our current view of the cytoskeletal architecture of the fission yeast microtubule comes from immunofluorescence microscopy and electron microscopy of static, fixed cells (2). However, recent technical advances in wide-field epifluorescence imaging of microtubules, made possible by fusions of green fluo- rescent protein to tubulin (GFP-tubulin), have allowed direct ob-

Columbia University, New York, New York 10032. 2

University of North Carolina, Chapel Hill, North Carolina 27514.

In the fission yeast, S. pombe, microtubules are required for multiple cellular processes, including maintenance of cell polarity, positioning of cellular organelles, and mitosis. Thus, microtubules are dynamic polymers (1), remodeling themselves within the living cell throughout the cell cycle.

Unfortunately, our current view of the cytoskeletal architecture of the fission yeast microtubule comes from immunofluorescence microscopy and electron microscopy of static, fixed cells (2). However, recent technical advances in wide-field epifluorescence imaging of microtubules, made possible by fusions of green fluo- rescent protein to tubulin (GFP-tubulin), have allowed direct ob-

Columbia University, New York, New York 10032. 2

University of North Carolina, Chapel Hill, North Carolina 27514.

servation of microtubule behavior in living fission yeast cells (3). We have now applied real-time confocal microscopy to GFP- tubulin in haploid fission yeast, and can report dynamic changes in the microtubule cytoskeleton with unprecedented spatial and tem- poral resolution.

A wild type haploid strain of fission yeast was transformed with a plasmid carrying the GFP-a2/tubulin gene. GFP-tubulin was therefore expressed along with endogenous tubulin. The behavior of the GFP-tubulin yeast strain is identical to that of the wild type in terms of cellular morphology and cell cycle duplication time (3). For imaging, cells were mounted on a thin layer of 20% gelatin mixed with yeast medium, between coverslip and slide. Images were digitally acquired at room temperature (23? to 26?C) with Metamorph Software (Universal Imaging Corp.) controlling a

servation of microtubule behavior in living fission yeast cells (3). We have now applied real-time confocal microscopy to GFP- tubulin in haploid fission yeast, and can report dynamic changes in the microtubule cytoskeleton with unprecedented spatial and tem- poral resolution.

A wild type haploid strain of fission yeast was transformed with a plasmid carrying the GFP-a2/tubulin gene. GFP-tubulin was therefore expressed along with endogenous tubulin. The behavior of the GFP-tubulin yeast strain is identical to that of the wild type in terms of cellular morphology and cell cycle duplication time (3). For imaging, cells were mounted on a thin layer of 20% gelatin mixed with yeast medium, between coverslip and slide. Images were digitally acquired at room temperature (23? to 26?C) with Metamorph Software (Universal Imaging Corp.) controlling a

A. A.

B. B.

Figure 1. Real-time confocal imaging of GFP-tubulin in the haploid fission yeast, S. pombe. Images are extracted at the noted time intervals to show cell cycle progression of microtubule reorganization and dynamics. Panel A shows an interphase microtubule bundle. Note the dynamic changes in microtubule lengths and the persistence of the overlap region; o = overlap region. Panel B shows a mitotic microtubule spindle. Note the rapid increase in spindle length at both ends, as well as astral microtubules and newly nucleated microtubules from the central region of the cell at the late stage of mitosis; s = spindle, a = astral microtubules. Scale bar = 5 ,um.

Figure 1. Real-time confocal imaging of GFP-tubulin in the haploid fission yeast, S. pombe. Images are extracted at the noted time intervals to show cell cycle progression of microtubule reorganization and dynamics. Panel A shows an interphase microtubule bundle. Note the dynamic changes in microtubule lengths and the persistence of the overlap region; o = overlap region. Panel B shows a mitotic microtubule spindle. Note the rapid increase in spindle length at both ends, as well as astral microtubules and newly nucleated microtubules from the central region of the cell at the late stage of mitosis; s = spindle, a = astral microtubules. Scale bar = 5 ,um.

262 262



Documents

Centennial Issue: October, 1899-1999 || Migration Forces in Dictyostelium Measured by Centrifuge DIC Microscopy