PubMed Health⌕ Search

Biomedical subjects

C E Walczak

Publications and source records attributed to C E Walczak.

At least 19 recordsLinked to original sources
PubMed2001

Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities.

The guanosine tri-phosphatase Ran stimulates assembly of microtubule spindles. However, it is not known what aspects of the microtubule cytoskeleton are subject to regulation by Ran in mitosis. Here we show that Ran-GTP stimulates microtubule assembly by increasing the rescue frequency of microtubules three- to eightfold. In addition to changing microtubule dynamics, Ran-GTP also alters the balance of motor activities, partly as a result of an increase in the amount of motile Eg5, a plus-end-directed microtubule motor that is essential for spindle formation. Thus, Ran regulates multiple processes that are involved in spindle assembly.

Animals↗
PubMed2000

Molecular mechanisms of spindle function.

The key molecules involved in regulating the assembly and function of the mitotic spindle are shared by evolutionarily divergent species. Studies in different model systems are leading to convergent conclusions about the central role of microtubule nucleation and dynamics and of kinesin-related motor proteins in spindle function.

Animals↗
PubMed2000

Microtubule dynamics and tubulin interacting proteins.

Microtubule dynamics are crucial in generation of the mitotic spindle. During the transition from interphase to mitosis, there is an increase in the frequency of microtubule catastrophes. Recent work has identified two proteins, Op 18/stathmin and XKCM1, which can promote microtubule catastrophes in vitro and in cells or extracts. Although both of these proteins share the ability to bind tubulin dimers, their mechanisms of action in destabilizing microtubules are distinct.

Animals↗
PubMed2000

Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts.

Microtubules are dynamic polymers that move stochastically between periods of growth and shrinkage, a property known as dynamic instability. Here, to investigate the mechanisms regulating microtubule dynamics in Xenopus egg extracts, we have cloned the complementary DNA encoding the microtubule-associated protein XMAP215 and investigated the function of the XMAP215 protein. Immunodepletion of XMAP215 indicated that it is a major microtubule-stabilizing factor in Xenopus egg extracts. During interphase, XMAP215 stabilizes microtubules primarily by opposing the activity of the destabilizing factor XKCM1, a member of the kinesin superfamily. These results indicate that microtubule dynamics in Xenopus egg extracts are regulated by a balance between a stabilizing factor, XMAP215, and a destabilizing factor, XKCM1.

Animals↗
PubMed1999

Microtubule dynamics in Xenopus egg extracts.

The organization and function of microtubules change dramatically during the cell cycle. At the onset of mitosis, a radial array of microtubules is broken down and reorganized into a bipolar spindle. This event requires changes in the dynamic behavior of individual microtubules. Through the use of Xenopus laevis egg extracts, a number of proteins affecting microtubule behavior have been identified. Recently, progress has also been made towards understanding how the activities of such microtubule-affecting proteins are regulated in a cell cycle-dependent manner. It is hoped that understanding how microtubule behavior is controlled during the cell cycle in vitro may illuminate the role of microtubule dynamics in various cellular processes.

Animals↗
PubMed1999

Kin I kinesins are microtubule-destabilizing enzymes.

Using in vitro assays with purified proteins, we show that XKCM1 and XKIF2, two distinct members of the internal catalytic domain (Kin I) kinesin subfamily, catalytically destabilize microtubules using a novel mechanism. Both XKCM1 and XKIF2 influence microtubule stability by targeting directly to microtubule ends where they induce a destabilizing conformational change. ATP hydrolysis recycles XKCM1/XKIF2 for multiple rounds of action by dissociating a XKCM1/ XKIF2-tubulin dimer complex released upon microtubule depolymerization. These results establish Kin I kinesins as microtubule-destabilizing enzymes, distinguish them mechanistically from kinesin superfamily members that use ATP hydrolysis to translocate along microtubules, and have important implications for the regulation of microtubule dynamics and for the intracellular functions and evolution of the kinesin superfamily.

Animals↗
PubMed1999

Microtubule-based motor function in mitosis.

Microtubule-based motors are essential both for the proper assembly of the mitotic spindle and for chromosome segregation. Mitotic motors in the yeast Saccharomyces cerevisiae exhibit either overlapping or opposing activities in order to achieve proper spindle function, whereas the analysis of motors using vertebrate cytoplasmic extracts has revealed less functional redundancy. In several systems, biochemical, genetic and two-hybrid approaches have been used both to identify associated nonmotor proteins and to address the molecular mechanisms behind kinetochore movements during chromosome alignment and segregation.

Animals↗
PubMed1997

A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts.

Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.

Animals↗
PubMed1997

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 alpha-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.

Amino Acid Sequence↗
PubMed1996

XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly.

We isolated a cDNA clone encoding a kinesin-related protein, which we named XKCM1. Antibodies to XKCM1 stain mitotic centromeres and spindle poles. Immunodepletion and antibody addition experiments in an in vitro spindle assembly assay show that XKCM1 is required for both establishment and maintenance of mitotic spindles. The structures that form in the absence of XKCM1 contain abnormally long microtubules. This long microtubule defect can be rescued by the addition of purified XKCM1 protein. Analysis of microtubule dynamics in a clarified mitotic extract reveals that loss of XKCM1 function causes a 4-fold suppression in the catastrophe frequency. XKCM1 thus exhibits a novel activity for a kinesin-related protein by promoting microtubule depolymerization during mitotic spindle assembly.

Amino Acid Sequence↗
PubMed1993

Identification of a family of casein kinases in Paramecium: biochemical characterization and cellular localization.

Protein phosphorylation is believed to play a role in the regulation of ciliary motility in the protozoan Paramecium tetraurelia. Five protein kinases from Paramecium, activated by cyclic nucleotides or Ca2+, have been characterized previously. We report here the identification of a family of second-messenger-independent casein kinases in Paramecium. Casein kinase activity was enriched in the soluble fraction of cilia, but there was also significant activity tightly associated with axonemes. Three ciliary casein kinase activities (soluble CKS1 and CKS2, and axonemal CKA) were separated by chromatography and characterized. The native forms of all three were monomeric, with molecular masses of 28-45 kDa as judged by in-gel kinase assays and sizing by gel filtration. CKS2 was inhibited by heparin, but CKA was unaffected and CKS1 was stimulated. All three activities preferred acidic substrates such as casein and phosvitin, but they could be distinguished by their preference for other substrates. Antibodies against mammalian casein kinase I recognized CKS1 and CKS2 in immunoblots (43 kDa), but did not stain CKA. The antibodies to casein kinase I were used to probe other cellular fractions. A 65 kDa antigen (particulate casein kinase, CKP) was enriched in particulate fractions of whole cells. This 65 kDa protein was found in isolated cell cortices, but was not present in the infraciliary lattice. This report represents the first biochemical identification of a casein kinase I family in protozoa.

Animals↗