Disruption of Four Kinesin Genes in Dictyostelium
© Nag et al; licensee BioMed Central Ltd. 2008
Received: 02 January 2008
Accepted: 22 April 2008
Published: 22 April 2008
Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans.
We performed individual disruptions of the kinesin genes, kif4, kif8, kif10, and kif11. None of the motors encoded by these genes are essential for development or viability of Dictyostelium. Removal of Kif4 (kinesin-7; CENP-E family) significantly impairs the rate of cell growth and, when combined with a previously characterized dynein inhibition, results in dramatic defects in mitotic spindle assembly. Kif8 (kinesin-4; chromokinesin family) and Kif10 (kinesin-8; Kip3 family) appear to cooperate with dynein to organize the interphase radial microtubule array.
The results reported here extend the number of kinesin gene disruptions in Dictyostelium, to now total 10, among the 13 isoforms. None of these motors, individually, are required for short-term viability. In contrast, homologs of at least six of the 10 kinesins are considered essential in humans. Our work underscores the functional redundancy of motor isoforms in basal organisms while highlighting motor specificity in more complex metazoans. Since motor disruption in Dictyostelium can readily be combined with other motility insults and stresses, this organism offers an excellent system to investigate functional interactions among the kinesin motor family.
Dictyostelium discoideum is a compact amoeba that spends much of its natural existence crawling through the soil, searching for and ingesting bacteria. When food sources are exhausted, individual amoebae trigger a developmental program that initiates both inter and intracellular signaling, to aggregate ~100,000 amoebae and form a multicellular mass. Each cell within this mass undergoes multiple adhesions and conformational changes, forming a cooperative slug that can migrate to new areas. The slug undergoes further multicellular differentiation to form supportive stalk cells, a rudimentary immuno-like surveillance system, and regenerative spores that resist environmental stresses. This dualistic life cycle and its associated transitions (single cell to metazoan organism) have made Dictyostelium an attractive model in which to study cell motility, signal transduction, and a relatively simple developmental program (reviewed in , see also ).
Motility-wise, Dictyostelium behaves in a manner similar to that of many vertebrate cells (crawling, sensing, and engulfing targets, robust intracellular movements). Yet, this organism clearly retains a simplicity associated with its relatively small and compact genome, and exhibits features commonly seen in protozoa and fungi (for example, an intranuclear spindle for cell division). Characterization of the actin cytoskeleton in Dictyostelium has led to the identification of actin binding proteins, multiple myosin motors, and signaling cascades whose functions are conserved among eukaryotic cells. Preliminary characterization of the microtubule-associated network has revealed a level of complexity intermediate between some of the simple single-celled eukaryotes and metazoans. For example, the machinery in Dictyostelium that drives movement along microtubules contains 14 motors (13 kinesin ATPases, 1 dynein ATPase, [3, 4]); twice the number found in Saccharomyces cerevisiae , but less than a quarter of the number encoded in the human genome . Paradoxically, deletions of kinesins whose homologs are essential for vertebrate activities have produced relatively mild phenotypes in Dictyostelium. Are these results reflective of Dictyostelium's unique life cycle? Or do they reveal core functional redundancies and interactions that, like the actin system work, can be utilized to understand microtubule-based motor action in more complex systems?
kif4, kif8, kif10, and kif11 are Not Essential Genes in Dictyostelium
Microtubule Distributions Appear Normal in Kinesin Null Cells
Kif8, Kif10, and Dynein Cooperate to Organize Interphase Microtubules
MT Array Morphology in Interphase Cells
Kif4 and Dynein Cooperate in Mitotic Spindle Assembly
We have presented gene deletions for four of the 13 kinesin family members in Dictyostelium, and have described the effects of these deletions on cell growth and viability. Individually, none of the four gene products is essential for cell viability nor do the proteins play critical roles in this organism's ability to undergo chemotaxis or to develop upon starvation. The knockout strains do, however, show subtle defects suggesting that many of the key forms of intracellular motility essential for Dictyostelium biosynthesis and reproduction are supported by more than one motor protein.
In wild-type Dictyostelium cells, both plus end-directed microtubule pushing, and minus end-directed pulling forces are important for maintenance of centrosome position and the radial distribution of interphase microtubules [18, 21]. If minus end-directed dynein motility is impaired, a kinesin-like activity appears to dominate and push both the centrosome and microtubule array throughout the cytoplasm . Here we have identified two kinesins, kif8 (kinesin-4 family) and kif10 (kinesin-8 family), that appear to collaborate with dynein in this organization process. In other eukaryotic cells, kinesin-4 motors participate in a number of diverse activities . One subset of kinesin-4 family members (KIF4) function during mitotic events, with chromatin- and spindle-associated motors that organize bipolar microtubule assemblies and facilitate chromosome alignment . Other subsets of kinesin-4 motors (e.g., KIF21) appear to power interphase organelle transport in cultured cells such as fibroblasts and post-mitotic neurons [24, 25]. The single Dictyostelium kinesin-4 (kif8) is a divergent member of this family, the motor domain is most closely homologous with KIF4 subfamily, yet it contains carboxy-terminal WD-40 repeat motifs in the heavy chain tail that are characteristic of the KIF21 subfamily [3, 22]. The kinesin-8 family of motors (kif10 in Dictyostelium) is thought to mediate chromosome movements through a combination of translocation and microtubule depolymerization activities (recently reviewed in , see also [27, 28]. The S. cerevisiae isoform (Kip3) has previously been shown to cooperate with dynein in positioning mitotic spindles through cortically mediated force production and through control of microtubule length [27, 29, 30]. Deletions of kinesin-8 isoforms in Schizosaccharomyces pombe also suggest a combined force and length control mechanism that positions nuclei and spindles through microtubule-cortex interactions [31, 32]. In the absence of either kinesin-4 or kinesin-8 in Dictyostelium, we are unable to induce the distinctive centrosome movements via dynein motor overexpression. It is conceivable that Kif8 and Kif10 counterbalance dynein-mediated forces through force-production or anchoring activities at the cell cortex (e.g. kinesin-8) and via lateral microtubule-microtubule interactions (e.g. kinesin-4) that supply sufficient rigidity to allow plus end-directed motors to effectively push (and not simply bend) microtubules. In wild-type Dictyostelium, the balance between opposing dynein and kinesin motor activities serves to reinforce the centrosome position and help maintain the radial character of the interphase microtubule array as these cells crawl around and change shape.
Disruption of the kinesin-7 motor (CENP-E) in the mouse is embryonic lethal ; this motor is thought to be essential for the proper connection between kinetochores of condensed chromosomes and the mitotic spindle . In contrast, neither member of the kinesin-7 family in Dictyostelium (Kif4, Kif11) is essential for mitosis, although removal of Kif4, the isoform that is most homologous to the vertebrate kinetochore CENP-E greatly affects cell growth rate. Preliminary characterization of Kif4 suggests that this motor functions together with dynein in organizing spindle assembly during cell division. While the motor domain of Kif11 is homologous with the kinesin-7 family , this polypeptide is significantly shorter and expressed at a much higher level than other CENP-E-like proteins. Outside of a minor enhancement of stationary phase cell density, removal of this motor has no obvious effect on cell viability or function. Closer inspection of each kinesin, and of cells lacking their expression will be required before we can fully understand their individual function(s)
Our study here extends previous work from several laboratories that, taken together, have individually deleted 10 of the total 13 kinesins in Dictyostelium [7, 9, 10, 12–14]. All of these deletions have proven to generate cell lines that can survive over multiple generations of growth, indicating that none of these 10 kinesin motors is immediately required for cell viability. Although the Kif12 disruption (kinesin-6, MKLP) produced significant defects in cytokinesis, mutant cells were still able to undergo some form of division that allows strain propagation . The only, potentially essential, kinesin gene reported so far in Dictyostelium encodes one of the organelle transporter motors, kif3 (kinesin-1 family). Kif3 can be isolated biochemically and shown capable of powering microtubule gliding, but efforts by Röhlk et al,  and in our own lab (Nag, Tikhonenko, and Koonce, unpublished) have not yet yielded viable cells lacking this motor. The resiliency of Dictyostelium to motor disruptions is similar to systematic analyses of kinesin isoforms in S. cerevisiae, where all six kinesin-related motors (and one dynein isoform) can be individually deleted without loss of viability . The yeast work provided a major guiding principle, for it was the first to suggest that high degree of functional redundancy is present among kinesin family members, and that deletion of motor combinations is required to inhibit cell division. Although, to our knowledge, complete survey disruptions have not yet been reported in other simple eukaryotes, there are clear indications of motor redundancy in some cell models such as S. pombe , Aspergillus nidulans, and Ustilago maydis . The kinesins in Dictyostelium likewise possess overlapping functions.
Mitotic kinesin disruptions in simple eukaryotes vs metazoans.
Kinesin- 8 (Kip3)
Kif2A, 2B, MCAK
Analysis of the kinesin gene family in Dictyostelium suggests that a significant level of functional redundancy or overlap exists among the organism's motor activities. This result is similar to findings from functional analyses performed in basal organisms such as yeast and fungi, but it contrasts sharply with the roles of individual motors in metazoans. At first glance, most of the kinesins in Dictyostelium can be deleted individually without penalty to growth or viability. Yet, upon closer scrutiny or in cases where we impose under additional stresses, we can discern clear phenotypic changes in the cell that provide insight into motor function that may not be obvious in other organisms. Given its greater complement of motor isoforms, and its greater utility of microtubule function relative to other basal eukaryotes, Dictyostelium offers an interesting model in which to investigate functional interactions and the regulation of multiple motor proteins.
Kinesin gene sequences were obtained from the dictybase website (see Availability and requirements section). The following primer combinations were used to amplify kinesin gene fragments from AX2 cell genomic DNA; also listed are the downstream kinesin gene-specific primers used for screening recombinants:
Forward: 5'CGCAAGCTT AGCCACCAAGACCATTACTTGGACCA 3' (-501 to -476)
Reverse: 5'CGCGAGCTC TTAAACTACCACCAATTATTGCGTCATT 3' (+1318 to +1345)
Screen: 5'CATCATCATCCTCTTCACCACTACTATT 3' (+1501 to +1528)
Forward: 5'CGCGGATCC GGGTTGCATTAAGAGTTAGACCC 3' (+44 to +66)
Reverse: 5'CCCAAGCTT GAATCGGCAGGACTAACACATGC 3' (+ 1302 to +1324)
Screen: 5'GATTGGTTAATACACACCTAATTG 3' (+1381 to +1404)
Forward 5'CGCGGATCC TGATCAATATGCAACTCAAGAAGAAG 3' (+249 to +274)
Reverse 5'CCCAAGCTT GATCATTGTCATCATCATCATC 3' (+1408 to +1429)
Screen: 5'GTATCATTGATTCATCATTATCCCT 3' (+1501 to +1525)
Forward: 5'CGCGGATCC GAATGAACGAGAATATATCGGTTAGC 3' (-2 to +24)
Reverse: 5'CCCAAGCTT CCATTACCACTACCACTACCACCT 3' (+1497 to +1520)
Screen: 5'TGACTTGGTGAAACAAATGTTGATC 3' (+1532 to +1556)
+1 of the numbering scheme refers to the position A of the ATG start codon. Restriction enzyme sites were engineered into the ends of each primer (BamH 1, Hind III or Sac 1, shown in bold type) to facilitate cloning of the amplified DNA into a pUC19 host plasmid, and (in most cases) to excise the DNA construct for transformation. Each construct was sequenced to confirm the identity of the kinesin fragment. Native restrictions sites (Fig. 2) were used to excise and replace an internal fragment of the kinesin sequences (47–669 bp) with a 1.6-kb blasticidin resistance cassette (Bsr r ) (Sma I digest) from pLRBLP , obtained from the Dictyostelium Stock Center (see Availability and requirements section for URL). Final constructs were again sequenced to determine the orientation of the Bsr r cassette (diagramed in Fig. 2). The kif8 construct was designed to terminate message coding at S202; kif10 at N223; kif11 at S151; and kif4 at W45. In all cases, these disruptions occur upstream of the microtubule-binding domain of the motor.
Standard molecular biology procedures were followed for DNA isolation, manipulation, and blotting. RNA was isolated using the RNeasy kit from Qiagen, following the manufacturer's instructions. kif8, kif10, and kif11 blots were probed with 32P-labeled DNA. the kif4 Southern blot was performed using chemiluminescence procedures (ECL, Amersham Biosciences). All blots (Southern and Northern) were probed with the initial amplified genomic target corresponding to the relevant kinesin clone, as indicated above and in Figure 2A.
A calcium phosphate procedure was used to transform Dictyostelium AX-2 cells, with 15 μg of linearized DNA per near confluent 10-cm dish (107 cells) . Transformants were selected with 5 μg/ml blasticidin. Individual colonies were picked with a pipette into 24 well plates, and were screened by PCR for homologous recombination. Amplification of a 1.6-kb target with a primer internal to the Bsr r marker (5' GAATGGCAAGTTAGTCAAAACTACG 3') and a primer downstream of the recombination site (indicated above for each kinesin sequence) was used to initially identify positive recombinants. Cells from positive colonies were further purified by serial dilution, and were again confirmed by PCR with downstream and upstream primer combinations. For dynein disruptions, we introduced a motor domain expression plasmid (aa 1384–4725), into kinesin null cells by either a CaPO4 or an electroporation method . kif-/380 K expressing cells were selected with 10 μg/ml G-418 (geneticin, Sigma Chemical Co).
Cells were flattened on glass coverslips using an agarose sheet, fixed with formaldehyde, labeled with a tubulin antibody , and in some cases Hoechst 33342, as described in . Z-series of images were obtained on a DeltaVision light microscopy workstation and were deconvolved using softWoRx 2.5 (Applied Precision, Issaquah, WA). Maximum intensity projections were compiled using ImageJ (NIH); figures were assembled in Adobe Photoshop. For cell growth measurements, triplicate 100-ml cultures were seeded with 9 × 104 cells/ml, shaken at 200 rpm at RT, and counted with a hemocytometer every 24 hr. Growth curves were calculated and displayed with Microsoft Excel; error bars indicate standard deviation.
Availability and requirements
We are grateful to the efforts at http://dictybase.org/ to archive and annotate Dictyostelium sequence information, and to the Dictyostelium Stock Center Resource for plasmids. Drs. Alexey Khodjakov and Conly Rieder provided valuable discussion and assistance with the light microscopy. We appreciate the use of Wadsworth Center's Molecular Genetics Core for DNA sequencing. This work was supported in part by the NSF (MCB-0542731 to MPK).
- Kessin RH: Dictyostelium. Evolution, Cell Biology, and the Development of Multicellularity. 2001, Cambridge University PressView ArticleGoogle Scholar
- Chen G, Zhuchenko O, Kuspa A: Immune-like Phagocyte Activity in the Social Amoeba. Science. 2007, 317 (5838): 678-681. 10.1126/science.1143991.PubMed CentralView ArticlePubMedGoogle Scholar
- Kollmar M, Glockner G: Identification and phylogenetic analysis of Dictyostelium discoideum kinesin proteins. BMC Genomics. 2003, 4 (1): 47-10.1186/1471-2164-4-47.PubMed CentralView ArticlePubMedGoogle Scholar
- Koonce MP, Grissom PM, McIntosh JR: Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein. J Cell Biol. 1992, 119 (6): 1597-1604. 10.1083/jcb.119.6.1597.View ArticlePubMedGoogle Scholar
- Hildebrandt ER, Hoyt MA: Mitotic motors in Saccharomyces cerevisiae. Biochim Biophys Acta (BBA) – Mol Cell Res. 2000, 1496 (1): 99-116. 10.1016/S0167-4889(00)00012-4.View ArticleGoogle Scholar
- Miki H, Setou M, Kaneshiro K, Hirokawa N: All kinesin superfamily protein, KIF, genes in mouse and human. Proc Nat Acad Sci (USA). 2001, 98 (13): 7004-7011. 10.1073/pnas.111145398.View ArticleGoogle Scholar
- Lakshmikanth GS, Warrick HM, Spudich JA: A mitotic kinesin-like protein required for normal karyokinesis, myosin localization to the furrow, and cytokinesis in Dictyostelium. Proc Natl Acad Sci USA. 2004, 101 (47): 16519-16524. 10.1073/pnas.0407304101.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Q, Lakshmikanth GS, Spudich JA, De Lozanne A: The Localization of Inner Centromeric Protein (INCENP) at the Cleavage Furrow Is Dependent on Kif12 and Involves Interactions of the N Terminus of INCENP with the Actin Cytoskeleton. Mol Biol Cell. 2007, 18 (9): 3366-3374. 10.1091/mbc.E06-10-0895.PubMed CentralView ArticlePubMedGoogle Scholar
- Pollock N, de Hostos EL, Turck CW, Vale RD: Reconstitution of membrane transport powered by a novel dimeric kinesin motor of the Unc104/KIF1A family purified from Dictyostelium. J Cell Biol. 1999, 147 (3): 493-506. 10.1083/jcb.147.3.493.PubMed CentralView ArticlePubMedGoogle Scholar
- Tikhonenko I, Nag DK, Martin N, Koonce MP: Kinesin-5 contributes to mitotic spindle stability in Dictyostelium. 2008, under reviewGoogle Scholar
- Koonce MP, Samsó M: Overexpression of cytoplasmic dynein's globular head causes a collapse of the interphase microtubule network in Dictyostelium. Mol Biol Cell. 1996, 7 (6): 935-948.PubMed CentralView ArticlePubMedGoogle Scholar
- de Hostos EL, McCaffrey G, Sucgang R, Pierce DW, Vale RD: A Developmentally Regulated Kinesin-related Motor Protein from Dictyostelium discoideum. Mol Biol Cell. 1998, 9 (8): 2093-2106.PubMed CentralView ArticlePubMedGoogle Scholar
- Iwai S, Ishiji A, Mabuchi I, Sutoh K: A Novel Actin-bundling Kinesin-related Protein from Dictyostelium discoideum. J Biol Chem. 2004, 279 (6): 4696-4704. 10.1074/jbc.M308022200.View ArticlePubMedGoogle Scholar
- Iwai S, Suyama E, Adachi H, Sutoh K: Characterization of a C-terminal-type kinesin-related protein from Dictyostelium discoideum. FEBS Letters. 2000, 475 (1): 47-51. 10.1016/S0014-5793(00)01619-7.View ArticlePubMedGoogle Scholar
- Zhu C, Zhao J, Bibikova M, Leverson JD, Bossy-Wetzel E, Fan JB, Abraham RT, Jiang W: Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. Mol Biol Cell. 2005, 16 (7): 3187-3199. 10.1091/mbc.E05-02-0167.PubMed CentralView ArticlePubMedGoogle Scholar
- Brown KD, Coulson RM, Yen TJ, Cleveland DW: Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis. J Cell Biol. 1994, 125 (6): 1303-1312. 10.1083/jcb.125.6.1303.View ArticlePubMedGoogle Scholar
- Yen TJ, Li G, Schaar BT, Szilak I, Cleveland DW: CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature. 1992, 359 (6395): 536-539. 10.1038/359536a0.View ArticlePubMedGoogle Scholar
- Koonce MP, Kohler J, Neujahr R, Schwartz JM, Tikhonenko I, Gerisch G: Dynein motor regulation stabilizes interphase microtubule arrays and determines centrosome position. EMBO J. 1999, 18 (23): 6786-6792. 10.1093/emboj/18.23.6786.PubMed CentralView ArticlePubMedGoogle Scholar
- Brito DA, Strauss J, Magidson V, Tikhonenko I, Khodjakov A, Koonce MP: Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium. Mol Biol Cell. 2005, 16 (7): 3334-3340. 10.1091/mbc.E05-01-0057.PubMed CentralView ArticlePubMedGoogle Scholar
- Koonce MP: Dictyostelium, a model organism for microtubule-based transport. Protist. 2000, 151 (1): 17-25. 10.1078/1434-4610-00004.View ArticlePubMedGoogle Scholar
- Koonce MP, Khodjakov A: Dynamic microtubules in Dictyostelium. J Muscle Res Cell Motil. 2002, 23 (7–8): 613-619. 10.1023/A:1024446821701.View ArticlePubMedGoogle Scholar
- Miki H, Okada Y, Hirokawa N: Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol. 2005, 15 (9): 467-476. 10.1016/j.tcb.2005.07.006.View ArticlePubMedGoogle Scholar
- Kurasawa Y, Earnshaw WC, Mochizuki Y, Dohmae N, Todokoro K: Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation. EMBO J. 2004, 23 (16): 3237-3248. 10.1038/sj.emboj.7600347.PubMed CentralView ArticlePubMedGoogle Scholar
- Midorikawa R, Takei Y, Hirokawa N: KIF4 Motor Regulates Activity-Dependent Neuronal Survival by Suppressing PARP-1 Enzymatic Activity. Cell. 2006, 125 (2): 371-383. 10.1016/j.cell.2006.02.039.View ArticlePubMedGoogle Scholar
- Sekine Y, Okada Y, Noda Y, Kondo S, Aizawa H, Takemura R, Hirokawa N: A novel microtubule-based motor protein (KIF4) for organelle transports, whose expression is regulated developmentally. J Cell Biol. 1994, 127 (1): 187-201. 10.1083/jcb.127.1.187.View ArticlePubMedGoogle Scholar
- Stumpff J, Wordeman L: Chromosome Congression: The Kinesin-8-Step Path to Alignment. Current Biology. 2007, 17 (9): R326-R328. 10.1016/j.cub.2007.03.013.PubMed CentralView ArticlePubMedGoogle Scholar
- Gupta ML, Carvalho P, Roof DM, Pellman D: Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle. Nat Cell Biol. 2006, 8 (9): 913-923. 10.1038/ncb1457.View ArticlePubMedGoogle Scholar
- Varga V, Helenius J, Tanaka K, Hyman AA, Tanaka TU, Howard J: Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner. Nat Cell Biol. 2006, 8 (9): 957-962. 10.1038/ncb1462.View ArticlePubMedGoogle Scholar
- Cottingham FR, Hoyt MA: Mitotic Spindle Positioning in Saccharomyces cerevisiae Is Accomplished by Antagonistically Acting Microtubule Motor Proteins. J Cell Biol. 1997, 138 (5): 1041-1053. 10.1083/jcb.138.5.1041.PubMed CentralView ArticlePubMedGoogle Scholar
- DeZwaan TM, Ellingson E, Pellman D, Roof DM: Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration. J Cell Biol. 1997, 138 (5): 1023-1040. 10.1083/jcb.138.5.1023.PubMed CentralView ArticlePubMedGoogle Scholar
- Tran PT, Marsh L, Doye V, Inoue S, Chang F: A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing. J Cell Biol. 2001, 153 (2): 397-412. 10.1083/jcb.153.2.397.PubMed CentralView ArticlePubMedGoogle Scholar
- West RR, Malmstrom T, Troxell CL, McIntosh JR: Two Related Kinesins, klp5+ and klp6+, Foster Microtubule Disassembly and Are Required for Meiosis in Fission Yeast. Mol Biol Cell. 2001, 12 (12): 3919-3932.PubMed CentralView ArticlePubMedGoogle Scholar
- Putkey FR, Cramer T, Morphew MK, Silk AD, Johnson RS, McIntosh JR, Cleveland DW: Unstable Kinetochore-Microtubule Capture and Chromosomal Instability Following Deletion of CENP-E. Developmental Cell. 2002, 3 (3): 351-365. 10.1016/S1534-5807(02)00255-1.View ArticlePubMedGoogle Scholar
- McEwen BF, Chan GKT, Zubrowski B, Savoian MS, Sauer MT, Yen TJ: CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can Be Achieved via Redundant Mechanisms in Mammalian Cells. Mol Biol Cell. 2001, 12 (9): 2776-2789.PubMed CentralView ArticlePubMedGoogle Scholar
- Rohlk C, Rohlfs M, Leier S, Schliwa M, Liu X, Parsch J, Woehlke G: Properties of the Kinesin-1 motor DdKif3 from Dictyostelium discoideum. European Journal of Cell Biology.Corrected Proof, ,Google Scholar
- Grishchuk EL, McIntosh JR: Microtubule depolymerization can drive poleward chromosome motion in fission yeast. EMBO J. 2006, 25 (20): 4888-4896. 10.1038/sj.emboj.7601353.PubMed CentralView ArticlePubMedGoogle Scholar
- Rischitor PE, Konzack S, Fischer R: The Kip3-Like Kinesin KipB Moves along Microtubules and Determines Spindle Position during Synchronized Mitoses in Aspergillus nidulans Hyphae. Eukaryotic Cell. 2004, 3 (3): 632-645. 10.1128/EC.3.3.632-645.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Schuchardt I, Assmann D, Thines E, Schuberth C, Steinberg G: Myosin-V, Kinesin-1, and Kinesin-3 Cooperate in Hyphal Growth of the Fungus Ustilago maydis. Mol Biol Cell. 2005, 16 (11): 5191-5201. 10.1091/mbc.E05-04-0272.PubMed CentralView ArticlePubMedGoogle Scholar
- Sonnichsen B, Koski LB, Walsh A, Marschall P, Neumann B, Brehm M, Alleaume AM, Artelt J, Bettencourt P, Cassin E: Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature. 2005, 434 (7032): 462-469. 10.1038/nature03353.View ArticlePubMedGoogle Scholar
- Goshima G, Vale RD: The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J Cell Biol. 2003, 162 (6): 1003-1016. 10.1083/jcb.200303022.PubMed CentralView ArticlePubMedGoogle Scholar
- Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GJ, Best AA, Cande WZ, Chen F, Cipriano MJ: Genomic Minimalism in the Early Diverging Intestinal Parasite Giardia lamblia. Science. 2007, 317 (5846): 1921-1926. 10.1126/science.1143837.View ArticlePubMedGoogle Scholar
- Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel AR: A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucl Acids Res. 2004, 32 (19): e143-10.1093/nar/gnh136.PubMed CentralView ArticlePubMedGoogle Scholar
- Egelhoff TT, Titus MA, Manstein DJ, Ruppel KM, Spudich JA: Molecular genetic tools for study of the cytoskeleton in Dictyostelium. Methods in Enzymology. Edited by: Vallee RB. 1991, Academic Press, 196: 319-334.Google Scholar
- Knecht DA, Jung J, Matthews L: Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells. Developmental Genetics. 1990, 11 (5–6): 403-409. 10.1002/dvg.1020110513.View ArticlePubMedGoogle Scholar
- Piperno G, Fuller MT: Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985, 101 (6): 2085-2094. 10.1083/jcb.101.6.2085.View ArticlePubMedGoogle Scholar
- Pollock N, Koonce MP, de Hostos EL, Vale RD: In vitro microtubule-based organelle transport in wild-type Dictyostelium and cells overexpressing a truncated dynein heavy chain. Cell Motil Cytoskeleton. 1998, 40 (3): 304-314. 10.1002/(SICI)1097-0169(1998)40:3<304::AID-CM8>3.0.CO;2-C.View ArticlePubMedGoogle Scholar
- Bishop JD, Han Z, Schumacher JM: The Caenorhabditis elegans Aurora B Kinase AIR-2 Phosphorylates and Is Required for the Localization of a BimC Kinesin to Meiotic and Mitotic Spindles. Mol Biol Cell. 2005, 16 (2): 742-756. 10.1091/mbc.E04-08-0682.PubMed CentralView ArticlePubMedGoogle Scholar
- Powers J, Bossinger O, Rose D, Strome S, Saxton W: A nematode kinesin required for cleavage furrow advancement. Current Biology. 1998, 8 (20): 1133-1136. 10.1016/S0960-9822(98)70470-1.PubMed CentralView ArticlePubMedGoogle Scholar
- Powers J, Rose DJ, Saunders A, Dunkelbarger S, Strome S, Saxton WM: Loss of KLP-19 polar ejection force causes misorientation and missegregation of holocentric chromosomes. J Cell Biol. 2004, 166 (7): 991-1001. 10.1083/jcb.200403036.PubMed CentralView ArticlePubMedGoogle Scholar
- Saunders AM, Powers J, Strome S, Saxton WM: Kinesin-5 acts as a brake in anaphase spindle elongation. Current Biology. 2007, 17 (12): R453-R454. 10.1016/j.cub.2007.05.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Segbert C, Barkus R, Powers J, Strome S, Saxton WM, Bossinger O: KLP-18, a Klp2 Kinesin, Is Required for Assembly of Acentrosomal Meiotic Spindles in Caenorhabditis elegans. Mol Biol Cell. 2003, 14 (11): 4458-4469. 10.1091/mbc.E03-05-0283.PubMed CentralView ArticlePubMedGoogle Scholar
- Manning AL, Ganem NJ, Bakhoum SF, Wagenbach M, Wordeman L, Compton DA: The Kinesin-13 Proteins Kif2a, Kif2b, and Kif2c/MCAK Have Distinct Roles during Mitosis in Human Cells. Mol Biol Cell. 2007, 18 (8): 2970-2979. 10.1091/mbc.E07-02-0110.PubMed CentralView ArticlePubMedGoogle Scholar
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