Apoptotic Cleavage of Cytoplasmic Dynein Intermediate Chain and P150GluedStops Dynein-Dependent Membrane Motility

Cytoplasmic dynein is the major minus end–directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150Glued. We have found that both CD-IC and p150Glued are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH2-terminal p150Glued binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150Glued in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein–driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein–dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

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3 NUCLEAR REPROGRAMMING OF PORCINE FIBROBLAST CELLS BY XENOPUS EGG EXTRACTS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab3 ◽

2006 ◽

Vol 18 (2) ◽

pp. 110 ◽

Cited By ~ 2

Author(s):

K. Miyamoto ◽

Y. Nagao ◽

N. Minami ◽

M. Yamada ◽

K. Ohsumi ◽

...

Keyword(s):

Mammalian Cells ◽

Marker Gene ◽

Calcium Ionophore ◽

Pcr Analysis ◽

Ionophore A23187 ◽

Fibroblast Cells ◽

Nuclear Reprogramming ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Much evidence indicates that somatic cells can be reprogrammed in an oocyte cytoplasm. The nuclear reprogramming consists of many unknown processes, and mechanisms underlying these processes still remain to be elucidated. Recently some reports noted that Xenopus oocytes or eggs can induce some of the reprogramming events in mammalian cells. We investigated the processes of nuclear reprogramming of porcine fibroblast cells by Xenopus egg extracts to understand how egg extracts trigger the reprogramming and/or dedifferentiation of cells. Unfertilized Xenopus eggs were collected from mature females. After removal of the jelly coat, activation was routinely achieved by calcium ionophore A23187. The eggs were immediately centrifuged and the cytoplasmic fraction was used as egg extracts. Porcine fibroblast cells were permeabilized by streptolysin O and incubated in the egg extracts under the ATP-generating system (1 mM ATP, 5 mM phosphocreatine, and 20 U/mL creatine kinase) for 30 min at 37�C or 2 h at 23�C. The incorporation of Xenopus-specific linker histone B4 into porcine fibroblasts was examined by immunofluorescence and immunobloting analysis. After collection of cells from the extracts, permeabilized membranes of the cells were resealed in culture medium containing 2 mM CaCl2 for 2 h. The cells were then incubated in DMEM with 10% fetal bovine serum (FBS) or porcine zygote medium-3 (PZM-3: Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) containing 5.55 mM glucose and 5% FBS. RNAs were extracted from the cells in each culture dish and Oct-4 expression was examined by RT-PCR analysis every day until Day 8. The primers were designed to span the 99 base-pair intron region of porcine Oct-4 gene for recognizing both spliced and unspliced transcripts. The incorporation of histone B4 from Xenopus egg extracts was observed at the nuclear region of the porcine fibroblasts under both the 37�C and the 23�C conditions. Because the histone B4 incorporation was inhibited by addition of Apyrase, an ATPase, a part of reprogramming might be an ATP-dependent process. When treated cells were incubated in DMEM or PZM-3, Oct-4 expression was detected in the cells cultured in DMEM, but not in PZM-3. However, the transcripts of Oct-4 were mainly obtained in unspliced form at the earlier stage of culture (after Day 1 to Day 4 of culture), suggesting that a part of reprogramming processes by the egg extracts involves induction of dedifferention of cells or activation of a pluripotent marker gene such as Oct-4. Xenopus egg extract may provide a system to investigate the processes involving nuclear reprogramming and the pluripotent state of mammalian cells in vitro.

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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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Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

Molecular Biology of the Cell ◽

10.1091/mbc.5.2.217 ◽

1994 ◽

Vol 5 (2) ◽

pp. 217-226 ◽

Cited By ~ 37

Author(s):

K E Sawin ◽

T J Mitchison

Keyword(s):

Basic Element ◽

Low Light ◽

Self Organized ◽

Egg Extracts ◽

Microtubule Motor ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Microtubule Motor Protein ◽

Poleward Flux

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.

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The Microtubule-destabilizing Kinesin XKCM1 Regulates Microtubule Dynamic Instability in Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e01-12-0143 ◽

2002 ◽

Vol 13 (8) ◽

pp. 2718-2731 ◽

Cited By ~ 112

Author(s):

Susan L. Kline-Smith ◽

Claire E. Walczak

Keyword(s):

Mammalian Cells ◽

Dynamic Instability ◽

Critical Role ◽

Dependent Manner ◽

Related Protein ◽

Microtubule Dynamic ◽

Mitotic Delay ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.

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Head and/or CaaX Domain Deletions of Lamin Proteins Disrupt Preformed Lamin A and C But Not Lamin B Structure in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.11.12.4323 ◽

2000 ◽

Vol 11 (12) ◽

pp. 4323-4337 ◽

Cited By ~ 56

Author(s):

Masako Izumi ◽

O. Anthony Vaughan ◽

Christopher J. Hutchison ◽

David M. Gilbert

Keyword(s):

Mammalian Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Soluble Form ◽

Lamin A ◽

Lamin B1 ◽

Head Domain ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A “head” domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, “head-less” lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.

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The Nup107-160 Nucleoporin Complex Is Required for Correct Bipolar Spindle Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1061 ◽

2006 ◽

Vol 17 (9) ◽

pp. 3806-3818 ◽

Cited By ~ 109

Author(s):

Arturo V. Orjalo ◽

Alexei Arnaoutov ◽

Zhouxin Shen ◽

Yekaterina Boyarchuk ◽

Samantha G. Zeitlin ◽

...

Keyword(s):

Mammalian Cells ◽

Spindle Checkpoint ◽

Spindle Assembly ◽

Sperm Chromatin ◽

Nuclear Pore ◽

Checkpoint Proteins ◽

Spindle Poles ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

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Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued.

The Journal of Cell Biology ◽

10.1083/jcb.131.6.1507 ◽

1995 ◽

Vol 131 (6) ◽

pp. 1507-1516 ◽

Cited By ~ 310

Author(s):

K T Vaughan ◽

R B Vallee

Keyword(s):

Amino Acids ◽

Rat Brain ◽

Direct Interaction ◽

Cytoplasmic Dynein ◽

Organelle Transport ◽

Cell Extracts ◽

Complex Protein ◽

Microtubule Motor ◽

Dynactin Complex ◽

Intermediate Chains

Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end-directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC-binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.

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Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

eLife ◽

10.7554/elife.53402 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Songli Zhu ◽

Mohammadjavad Paydar ◽

Feifei Wang ◽

Yanqiu Li ◽

Ling Wang ◽

...

Keyword(s):

Dna Damage ◽

Mammalian Cells ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

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Membrane/microtubule tip attachment complexes (TACs) allow the assembly dynamics of plus ends to push and pull membranes into tubulovesicular networks in interphase Xenopus egg extracts.

The Journal of Cell Biology ◽

10.1083/jcb.130.5.1161 ◽

1995 ◽

Vol 130 (5) ◽

pp. 1161-1169 ◽

Cited By ~ 75

Author(s):

C M Waterman-Storer ◽

J Gregory ◽

S F Parsons ◽

E D Salmon

Keyword(s):

Dynamic Instability ◽

Atp Depletion ◽

Immunofluorescent Staining ◽

Egg Extracts ◽

Microtubule Motor ◽

Xenopus Egg ◽

Membrane Tubules ◽

Membrane Networks ◽

Xenopus Egg Extracts ◽

Push And Pull

We discovered by using high resolution video microscopy, that membranes become attached selectively to the growing plus ends of microtubules by membrane/microtubule tip attachment complexes (TACs) in interphase-arrested, undiluted, Xenopus egg extracts. Persistent plus end growth of stationary microtubules pushed the membranes into thin tubules and dragged them through the cytoplasm at the approximately 20 microns/min velocity typical of free plus ends. Membrane tubules also remained attached to plus ends when they switched to the shortening phase of dynamic instability at velocities typical of free ends, 50-60 microns/min. Over time, the membrane tubules contacted and fused with one another along their lengths, forming a polygonal network much like the distribution of ER in cells. Several components of the membrane networks formed by TACs were identified as ER by immunofluorescent staining using antibodies to ER-resident proteins. TAC motility was not inhibited by known inhibitors of microtubule motor activity, including 5 mM AMP-PNP, 250 microM orthovanadate, and ATP depletion. These results show that membrane/microtubule TACs enable polymerizing ends to push and depolymerizing ends to pull membranes into thin tubular extensions and networks at fast velocities.

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A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111416657 ◽

2011 ◽

Vol 16 (9) ◽

pp. 995-1006 ◽

Cited By ~ 13

Author(s):

Curtis A. Thorne ◽

Bonnie Lafleur ◽

Michelle Lewis ◽

Alison J. Hanson ◽

Kristin K. Jernigan ◽

...

Keyword(s):

High Throughput Screening ◽

Mammalian Cells ◽

Wnt Pathway ◽

Renilla Luciferase ◽

Signal Distribution ◽

Egg Extracts ◽

Bona Fide ◽

Xenopus Egg ◽

The Stability ◽

Xenopus Egg Extracts

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

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