XCTK2: A Kinesin-related Protein That Promotes Mitotic Spindle Assembly in Xenopus laevis Egg Extracts

We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central α-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor- arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.

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Chromokinesin Xklp1 Contributes to the Regulation of Microtubule Density and Organization during Spindle Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e05-04-0271 ◽

2006 ◽

Vol 17 (3) ◽

pp. 1451-1460 ◽

Cited By ~ 20

Author(s):

Mirco Castoldi ◽

Isabelle Vernos

Keyword(s):

Cell Division ◽

Spindle Assembly ◽

Embryonic Cell ◽

Microtubule Polymerization ◽

M Phase ◽

Egg Extracts ◽

Xenopus Egg ◽

Directed Motility ◽

Xenopus Egg Extracts ◽

Bipolar Spindles

Xklp1 is a chromosome-associated kinesin required for Xenopus early embryonic cell division. Function blocking experiments in Xenopus egg extracts suggested that it is required for spindle assembly. We have reinvestigated Xklp1 function(s) by monitoring spindle assembly and microtubule behavior under a range of Xklp1 concentrations in egg extracts. We found that in the absence of Xklp1, bipolar spindles form with a reduced efficiency and display abnormalities associated with an increased microtubule mass. Likewise, centrosomal asters assembled in Xklp1-depleted extract show an increased microtubule mass. Conversely, addition of recombinant Xklp1 to the extract reduces the microtubule mass associated with spindles and asters. Our data suggest that Xklp1 affects microtubule polymerization during M-phase. We propose that these attributes, combined with Xklp1 plus-end directed motility, contribute to the assembly of a functional bipolar spindle.

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Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly in Xenopus Egg Extracts

Molecular Biology of the Cell ◽

10.1091/mbc.e04-05-0385 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5318-5328 ◽

Cited By ~ 68

Author(s):

Stéphane Brunet ◽

Teresa Sardon ◽

Timo Zimmerman ◽

Torsten Wittmann ◽

Rainer Pepperkok ◽

...

Keyword(s):

Spindle Assembly ◽

Aurora A Kinase ◽

Aurora A ◽

Microtubule Nucleation ◽

Terminal Domain ◽

Large N ◽

Egg Extracts ◽

Xenopus Egg ◽

Domain Functional ◽

Xenopus Egg Extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.

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MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts

Journal of Cell Science ◽

10.1242/jcs.109.1.239 ◽

1996 ◽

Vol 109 (1) ◽

pp. 239-246 ◽

Cited By ~ 10

Author(s):

A. Abrieu ◽

T. Lorca ◽

J.C. Labbe ◽

N. Morin ◽

S. Keyse ◽

...

Keyword(s):

Map Kinase ◽

Degradation Pathway ◽

Cdc2 Kinase ◽

Vitro Assay ◽

Kinase Activation ◽

Egg Extracts ◽

Dependent Protein ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.

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Peroxisome protein import recapitulated in Xenopus egg extracts

The Journal of Cell Biology ◽

10.1083/jcb.201901152 ◽

2019 ◽

Vol 218 (6) ◽

pp. 2021-2034 ◽

Cited By ~ 3

Author(s):

Fabian B. Romano ◽

Neil B. Blok ◽

Tom A. Rapoport

Keyword(s):

Strong Evidence ◽

Protein Import ◽

Atp Hydrolysis ◽

In Vitro Assay ◽

Vitro Assay ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Import System

Peroxisomes import their luminal proteins from the cytosol. Most substrates contain a C-terminal Ser-Lys-Leu (SKL) sequence that is recognized by the receptor Pex5. Pex5 binds to peroxisomes via a docking complex containing Pex14, and recycles back into the cytosol following its mono-ubiquitination at a conserved Cys residue. The mechanism of peroxisome protein import remains incompletely understood. Here, we developed an in vitro import system based on Xenopus egg extracts. Import is dependent on the SKL motif in the substrate and on the presence of Pex5 and Pex14, and is sustained by ATP hydrolysis. A protein lacking an SKL sequence can be coimported, providing strong evidence for import of a folded protein. The conserved cysteine in Pex5 is not essential for import or to clear import sites for subsequent rounds of translocation. This new in vitro assay will be useful for further dissecting the mechanism of peroxisome protein import.

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Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts

Nature ◽

10.1038/382420a0 ◽

1996 ◽

Vol 382 (6590) ◽

pp. 420-425 ◽

Cited By ~ 661

Author(s):

Rebecca Heald ◽

Régis Tournebize ◽

Thiemo Blank ◽

Raphael Sandaltzopoulos ◽

Peter Becker ◽

...

Keyword(s):

Self Organization ◽

Artificial Chromosomes ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Bipolar Spindles

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A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle

The Journal of Cell Biology ◽

10.1083/jcb.200304144 ◽

2003 ◽

Vol 161 (6) ◽

pp. 1021-1028 ◽

Cited By ~ 39

Author(s):

Melinda M. Horne ◽

Thomas M. Guadagno

Keyword(s):

Map Kinase ◽

Mitotic Spindle ◽

Kinase Inhibitor ◽

Map Kinase Phosphatase ◽

Egg Extracts ◽

Xenopus Egg ◽

Multiple Spindle ◽

Xenopus Egg Extracts ◽

Nih 3T3 ◽

Bipolar Spindles

Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.

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Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization

The Journal of Cell Biology ◽

10.1083/jcb.138.3.615 ◽

1997 ◽

Vol 138 (3) ◽

pp. 615-628 ◽

Cited By ~ 248

Author(s):

Rebecca Heald ◽

Régis Tournebize ◽

Anja Habermann ◽

Eric Karsenti ◽

Anthony Hyman

Keyword(s):

Spindle Assembly ◽

Cytoplasmic Dynein ◽

Microtubule Organization ◽

Spindle Poles ◽

Egg Extracts ◽

Xenopus Egg ◽

Sperm Nuclei ◽

Xenopus Egg Extracts ◽

Microtubule Stabilizing Agent ◽

Mitotic Chromatin

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end–directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

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Lamin B receptor-mediated chromatin tethering to the nuclear envelope is detrimental to the xenopus blastula

The Journal of Biochemistry ◽

10.1093/jb/mvaa123 ◽

2020 ◽

Author(s):

Haruka Oda ◽

Satsuki Kato ◽

Keita Ohsumi ◽

Mari Iwabuchi

Keyword(s):

Nuclear Envelope ◽

Mitotic Spindles ◽

Filamentous Actin ◽

Blastula Stage ◽

Lamin B ◽

Lamin B Receptor ◽

Specific Manner ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Abstract In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear laminar architecture and nuclear morphology, and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.

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