Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Subscribe

Logo for

Science 310 (5746): 310-314

Copyright © 2005 by the American Association for the Advancement of Science

Counting Cytokinesis Proteins Globally and Locally in Fission Yeast

Jian-Qiu Wu1, and Thomas D. Pollard1,2*

Abstract: We used fluorescence microscopy to measure global and local concentrations of 28 cytoskeletal and signaling proteins fused to yellow fluorescent protein (YFP) in the fission yeast Schizosaccharomyces pombe. Native promoters controlled the expression of these functional YFP fusion proteins. Fluorescence measured by microscopy or flow cytometry was directly proportional to protein concentration measured by quantitative immunoblotting. Global cytoplasmic concentrations ranged from 0.04 (formin Cdc12p) to 63 micromolar (actin). Proteins concentrated up to 100 times in contractile rings and 7500 times in spindle pole bodies at certain times in the cell cycle. This approach can be used to measure the global and local concentrations of any fusion protein.

1 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520–8103, USA.
2 Departments of Cell Biology and Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520–8103, USA.

* To whom correspondence should be addressed. E-mail: thomas.pollard{at}yale.edu

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Blt1 and Mid1 Provide Overlapping Membrane Anchors To Position the Division Plane in Fission Yeast.
M. Guzman-Vendrell, S. Baldissard, M. Almonacid, A. Mayeux, A. Paoletti, and J. B. Moseley (2013)
Mol. Cell. Biol. 33, 418-428
   Abstract »    Full Text »    PDF »
Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast.
J. Huang, Y. Huang, H. Yu, D. Subramanian, A. Padmanabhan, R. Thadani, Y. Tao, X. Tang, R. Wedlich-Soldner, and M. K. Balasubramanian (2012)
J. Cell Biol. 199, 831-847
   Abstract »    Full Text »    PDF »
Characterization of structural and functional domains of the anillin-related protein Mid1p that contribute to cytokinesis in fission yeast.
S. Saha and T. D. Pollard (2012)
Mol. Biol. Cell 23, 3993-4007
   Abstract »    Full Text »    PDF »
Cylindrical cellular geometry ensures fidelity of division site placement in fission yeast.
M. Mishra, Y. Huang, P. Srivastava, R. Srinivasan, M. Sevugan, R. Shlomovitz, N. Gov, M. Rao, and M. Balasubramanian (2012)
J. Cell Sci. 125, 3850-3857
   Abstract »    Full Text »    PDF »
{alpha}-Actinin and fimbrin cooperate with myosin II to organize actomyosin bundles during contractile-ring assembly.
D. Laporte, N. Ojkic, D. Vavylonis, and J.-Q. Wu (2012)
Mol. Biol. Cell 23, 3094-3110
   Abstract »    Full Text »    PDF »
Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase.
J. Al-Bassam, H. Kim, I. Flor-Parra, N. Lal, H. Velji, and F. Chang (2012)
Mol. Biol. Cell 23, 2878-2890
   Abstract »    Full Text »    PDF »
Oscillatory Dynamics of Cdc42 GTPase in the Control of Polarized Growth.
M. Das, T. Drake, D. J. Wiley, P. Buchwald, D. Vavylonis, and F. Verde (2012)
Science 337, 239-243
   Abstract »    Full Text »    PDF »
Characterization of Mid1 domains for targeting and scaffolding in fission yeast cytokinesis.
I.-J. Lee and J.-Q. Wu (2012)
J. Cell Sci. 125, 2973-2985
   Abstract »    Full Text »    PDF »
Subpixel colocalization reveals amyloid precursor protein-dependent kinesin-1 and dynein association with axonal vesicles.
L. Szpankowski, S. E. Encalada, and L. S. B. Goldstein (2012)
PNAS 109, 8582-8587
   Abstract »    Full Text »    PDF »
Stoichiometry of Nck-dependent actin polymerization in living cells.
J. A. Ditlev, P. J. Michalski, G. Huber, G. M. Rivera, W. A. Mohler, L. M. Loew, and B. J. Mayer (2012)
J. Cell Biol. 197, 643-658
   Abstract »    Full Text »    PDF »
An extended {gamma}-tubulin ring functions as a stable platform in microtubule nucleation.
S. Erlemann, A. Neuner, L. Gombos, R. Gibeaux, C. Antony, and E. Schiebel (2012)
J. Cell Biol. 197, 59-74
   Abstract »    Full Text »    PDF »
Roles of putative Rho-GEF Gef2 in division-site positioning and contractile-ring function in fission yeast cytokinesis.
Y. Ye, I.-J. Lee, K. W. Runge, and J.-Q. Wu (2012)
Mol. Biol. Cell 23, 1181-1195
   Abstract »    Full Text »    PDF »
Meiotic actin rings are essential for proper sporulation in fission yeast.
H. Yan and M. K. Balasubramanian (2012)
J. Cell Sci. 125, 1429-1439
   Abstract »    Full Text »    PDF »
Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells.
J. Yi, X. S. Wu, T. Crites, and J. A. Hammer III (2012)
Mol. Biol. Cell 23, 834-852
   Abstract »    Full Text »    PDF »
Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range.
T. M. Thomson, K. R. Benjamin, A. Bush, T. Love, D. Pincus, O. Resnekov, R. C. Yu, A. Gordon, A. Colman-Lerner, D. Endy, et al. (2011)
PNAS 108, 20265-20270
   Abstract »    Full Text »    PDF »
Myosin concentration underlies cell size-dependent scalability of actomyosin ring constriction.
M. E. K. Calvert, G. D. Wright, F. Y. Leong, K.-H. Chiam, Y. Chen, G. Jedd, and M. K. Balasubramanian (2011)
J. Cell Biol. 195, 799-813
   Abstract »    Full Text »    PDF »
CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast.
V. C. Coffman, P. Wu, M. R. Parthun, and J.-Q. Wu (2011)
J. Cell Biol. 195, 563-572
   Abstract »    Full Text »    PDF »
Gelsolin and Non-muscle Myosin IIA Interact to Mediate Calcium-regulated Collagen Phagocytosis.
P. D. Arora, Y. Wang, P. A. Janmey, A. Bresnick, H. L. Yin, and C. A. McCulloch (2011)
J. Biol. Chem. 286, 34184-34198
   Abstract »    Full Text »    PDF »
Quantification of the Brassinosteroid Insensitive1 Receptor in Planta.
G. Wilma van Esse, A. H. Westphal, R. P. Surendran, C. Albrecht, B. van Veen, J. W. Borst, and S. C. de Vries (2011)
Plant Physiology 156, 1691-1700
   Abstract »    Full Text »    PDF »
Quantitative dissection of the simple repression input-output function.
H. G. Garcia and R. Phillips (2011)
PNAS 108, 12173-12178
   Abstract »    Full Text »    PDF »
Assembly and architecture of precursor nodes during fission yeast cytokinesis.
D. Laporte, V. C. Coffman, I.-J. Lee, and J.-Q. Wu (2011)
J. Cell Biol. 192, 1005-1021
   Abstract »    Full Text »    PDF »
Purification of Actin from Fission Yeast Schizosaccharomyces pombe and Characterization of Functional Differences from Muscle Actin.
S.-C. Ti and T. D. Pollard (2011)
J. Biol. Chem. 286, 5784-5792
   Abstract »    Full Text »    PDF »
pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation.
M. An, D. Wijesinghe, O. A. Andreev, Y. K. Reshetnyak, and D. M. Engelman (2010)
PNAS 107, 20246-20250
   Abstract »    Full Text »    PDF »
Arabidopsis FIMBRIN5, an Actin Bundling Factor, Is Required for Pollen Germination and Pollen Tube Growth.
Y. Wu, J. Yan, R. Zhang, X. Qu, S. Ren, N. Chen, and S. Huang (2010)
PLANT CELL 22, 3745-3763
   Abstract »    Full Text »    PDF »
Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe.
R. Cabrera, Z. Sha, T. J. Vadakkan, J. Otero, F. Kriegenburg, R. Hartmann-Petersen, M. E. Dickinson, and E. C. Chang (2010)
Mol. Biol. Cell 21, 3125-3136
   Abstract »    Full Text »    PDF »
Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast.
V. Sirotkin, J. Berro, K. Macmillan, L. Zhao, and T. D. Pollard (2010)
Mol. Biol. Cell 21, 2894-2904
   Abstract »    Full Text »    PDF »
Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments.
J. Berro, V. Sirotkin, and T. D. Pollard (2010)
Mol. Biol. Cell 21, 2905-2915
   Abstract »    Full Text »    PDF »
Post-Golgi Sec Proteins Are Required for Autophagy in Saccharomyces cerevisiae.
J. Geng, U. Nair, K. Yasumura-Yorimitsu, and D. J. Klionsky (2010)
Mol. Biol. Cell 21, 2257-2269
   Abstract »    Full Text »    PDF »
Stoichiometry and Architecture of Active DNA Replication Machinery in Escherichia coli.
R. Reyes-Lamothe, D. J. Sherratt, and M. C. Leake (2010)
Science 328, 498-501
   Abstract »    Full Text »    PDF »
F-Actin Dynamics in Neurospora crassa.
A. Berepiki, A. Lichius, J. Y. Shoji, J. Tilsner, and N. D. Read (2010)
Eukaryot. Cell 9, 547-557
   Abstract »    Full Text »    PDF »
Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast.
B. C. Stark, T. E. Sladewski, L. W. Pollard, and M. Lord (2010)
Mol. Biol. Cell 21, 989-1000
   Abstract »    Full Text »    PDF »
The transcription factor Foxg1 regulates the competence of telencephalic cells to adopt subpallial fates in mice.
M. Manuel, B. Martynoga, T. Yu, J. D. West, J. O. Mason, and D. J. Price (2010)
Development 137, 487-497
   Abstract »    Full Text »    PDF »
In Vivo Stoichiometry of Shelterin Components.
K. K. Takai, S. Hooper, S. Blackwood, R. Gandhi, and T. de Lange (2010)
J. Biol. Chem. 285, 1457-1467
   Abstract »    Full Text »    PDF »
A feeling for the numbers in biology.
R. Phillips and R. Milo (2009)
PNAS 106, 21465-21471
   Abstract »    Full Text »    PDF »
Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis.
V. C. Coffman, A. H. Nile, I-J. Lee, H. Liu, and J.-Q. Wu (2009)
Mol. Biol. Cell 20, 5195-5210
   Abstract »    Full Text »    PDF »
The Association of the Arabidopsis Actin-Related Protein2/3 Complex with Cell Membranes Is Linked to Its Assembly Status But Not Its Activation.
S. O. Kotchoni, T. Zakharova, E. L. Mallery, J. Le, S. E.-D. El-Assal, and D. B. Szymanski (2009)
Plant Physiology 151, 2095-2109
   Abstract »    Full Text »    PDF »
Cellular Symmetry Breaking during Caenorhabditis elegans Development.
E. Munro and B. Bowerman (2009)
Cold Spring Harb Perspect Biol 1, a003400
   Abstract »    Full Text »    PDF »
Accuracy and precision in quantitative fluorescence microscopy.
J. C. Waters (2009)
J. Cell Biol. 185, 1135-1148
   Abstract »    Full Text »    PDF »
Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast.
C. T. Skau, E. M. Neidt, and D. R. Kovar (2009)
Mol. Biol. Cell 20, 2160-2173
   Abstract »    Full Text »    PDF »
Stepping into the ring: the SIN takes on contractile ring assembly.
R. H. Roberts-Galbraith and K. L. Gould (2008)
Genes & Dev. 22, 3082-3088
   Abstract »    Full Text »    PDF »
Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy.
J. Geng, M. Baba, U. Nair, and D. J. Klionsky (2008)
J. Cell Biol. 182, 129-140
   Abstract »    Full Text »    PDF »
G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers.
C. Higashida, S. Suetsugu, T. Tsuji, J. Monypenny, S. Narumiya, and N. Watanabe (2008)
J. Cell Sci. 121, 3403-3412
   Abstract »    Full Text »    PDF »
Biochemical and Structural Characterization of the Pak1-LC8 Interaction.
C. M. Lightcap, S. Sun, J. D. Lear, U. Rodeck, T. Polenova, and J. C. Williams (2008)
J. Biol. Chem. 283, 27314-27324
   Abstract »    Full Text »    PDF »
Structure and Biochemical Properties of Fission Yeast Arp2/3 Complex Lacking the Arp2 Subunit.
B. J. Nolen and T. D. Pollard (2008)
J. Biol. Chem. 283, 26490-26498
   Abstract »    Full Text »    PDF »
The Meiosis-Specific Sid2p-related Protein Slk1p Regulates Forespore Membrane Assembly in Fission Yeast.
H. Yan, W. Ge, T. G. Chew, J. Y. Chow, D. McCollum, A. M. Neiman, and M. K. Balasubramanian (2008)
Mol. Biol. Cell 19, 3676-3690
   Abstract »    Full Text »    PDF »
The Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus Inhibits Cell Cytokinesis and Proliferation by Interacting with Translation Elongation Factor 1{alpha}.
B. Zhou, J. Liu, Q. Wang, X. Liu, X. Li, P. Li, Q. Ma, and C. Cao (2008)
J. Virol. 82, 6962-6971
   Abstract »    Full Text »    PDF »
Yeast UCS proteins promote actomyosin interactions and limit myosin turnover in cells.
M. Lord, T. E. Sladewski, and T. D. Pollard (2008)
PNAS 105, 8014-8019
   Abstract »    Full Text »    PDF »
Regulation and Targeting of the Fission Yeast Formin cdc12p in Cytokinesis.
A. Yonetani, R. J. Lustig, J. B. Moseley, T. Takeda, B. L. Goode, and F. Chang (2008)
Mol. Biol. Cell 19, 2208-2219
   Abstract »    Full Text »    PDF »
The Clp1/Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1.
D. M. Clifford, B. A. Wolfe, R. H. Roberts-Galbraith, W. H. McDonald, J. R. Yates III, and K. L. Gould (2008)
J. Cell Biol. 181, 79-88
   Abstract »    Full Text »    PDF »
Counteractive Control of Polarized Morphogenesis during Mating by Mitogen-activated Protein Kinase Fus3 and G1 Cyclin-dependent Kinase.
L. Yu, M. Qi, M. A. Sheff, and E. A. Elion (2008)
Mol. Biol. Cell 19, 1739-1752
   Abstract »    Full Text »    PDF »
Counting Functional Inositol 1,4,5-Trisphosphate Receptors into the Plasma Membrane.
O. Dellis, A. M. Rossi, S. G. Dedos, and C. W. Taylor (2008)
J. Biol. Chem. 283, 751-755
   Abstract »    Full Text »    PDF »
Assembly Mechanism of the Contractile Ring for Cytokinesis by Fission Yeast.
D. Vavylonis, J.-Q. Wu, S. Hao, B. O'Shaughnessy, and T. D. Pollard (2008)
Science 319, 97-100
   Abstract »    Full Text »    PDF »
Intrinsic Dynamic Behavior of Fascin in Filopodia.
Y. S. Aratyn, T. E. Schaus, E. W. Taylor, and G. G. Borisy (2007)
Mol. Biol. Cell 18, 3928-3940
   Abstract »    Full Text »    PDF »
Single Molecule Kinetic Analysis of Actin Filament Capping: POLYPHOSPHOINOSITIDES DO NOT DISSOCIATE CAPPING PROTEINS.
J. R. Kuhn and T. D. Pollard (2007)
J. Biol. Chem. 282, 28014-28024
   Abstract »    Full Text »    PDF »
Rational design of memory in eukaryotic cells.
C. M. Ajo-Franklin, D. A. Drubin, J. A. Eskin, E. P.S. Gee, D. Landgraf, I. Phillips, and P. A. Silver (2007)
Genes & Dev. 21, 2271-2276
   Abstract »    Full Text »    PDF »
Three-dimensional arrangement of F-actin in the contractile ring of fission yeast.
T. Kamasaki, M. Osumi, and I. Mabuchi (2007)
J. Cell Biol. 178, 765-771
   Abstract »    Full Text »    PDF »
Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin.
F. Chaudhry, C. Guerin, M. von Witsch, L. Blanchoin, and C. J. Staiger (2007)
Mol. Biol. Cell 18, 3002-3014
   Abstract »    Full Text »    PDF »
Counting Low-Copy Number Proteins in a Single Cell.
B. Huang, H. Wu, D. Bhaya, A. Grossman, S. Granier, B. K. Kobilka, and R. N. Zare (2007)
Science 315, 81-84
   Abstract »    Full Text »    PDF »
The Yeast Actin Cytoskeleton: from Cellular Function to Biochemical Mechanism.
J. B. Moseley and B. L. Goode (2006)
Microbiol. Mol. Biol. Rev. 70, 605-645
   Abstract »    Full Text »    PDF »
A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase-Anaphase Transition in Sea Urchin Eggs.
A. Lucero, C. Stack, A. R. Bresnick, and C. B. Shuster (2006)
Mol. Biol. Cell 17, 4093-4104
   Abstract »    Full Text »    PDF »
Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast.
J.-Q. Wu, V. Sirotkin, D. R. Kovar, M. Lord, C. C. Beltzner, J. R. Kuhn, and T. D. Pollard (2006)
J. Cell Biol. 174, 391-402
   Abstract »    Full Text »    PDF »
The fission yeast Chs2 protein interacts with the type-II myosin Myo3p and is required for the integrity of the actomyosin ring.
R. Martin-Garcia and M.-H. Valdivieso (2006)
J. Cell Sci. 119, 2768-2779
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882