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T J Mitchison

Publications and source records attributed to T J Mitchison.

At least 55 records · Page 3Linked to original sources
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

The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

Actin Cytoskeleton↗
PubMed1997

Identification of novel graded polarity actin filament bundles in locomoting heart fibroblasts: implications for the generation of motile force.

We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25-2.5 microm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 microm, but can reach up to about 30 microm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward. By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body. This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.

Actin Cytoskeleton↗
PubMed1997

Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails.

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.

Actin Cytoskeleton↗
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↗
PubMed1997

Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes.

The pathogenic bacterium Listeria monocytogenes is capable of directed movement within the cytoplasm of infected host cells. Propulsion is thought to be driven by actin polymerization at the bacterial cell surface, and moving bacteria leave in their wake a tail of actin filaments. Determining the mechanism by which L. monocytogenes polymerizes actin may aid the understanding of how actin polymerization is controlled in the cell. Actin assembly by L. monocytogenes requires the bacterial surface protein ActA and protein components present in host cell cytoplasm. We have purified an eight-polypeptide complex that possesses the properties of the host-cell actin polymerization factor. The pure complex is sufficient to initiate ActA-dependent actin polymerization at the surface of L. monocytogenes, and is required to mediate actin tail formation and motility. Two subunits of this protein complex are actin-related proteins (ARPs) belonging to the Arp2 and Arp3 subfamilies. The Arp3 subunit localizes to the surface of stationary bacteria and the tails of motile bacteria in tissue culture cells infected with L. monocytogenes; this is consistent with a role for the complex in promoting actin assembly in vivo. The activity and subunit composition of the Arp2/3 complex suggests that it forms a template that nucleates actin polymerization.

Actin-Related Protein 2↗
PubMed1997

Actin dynamics in vivo.

Actin dynamics in lamellipodia are driven by continuous cycles of actin polymerization, retrograde flow, and depolymerization. In the past year, advances have been made in identifying signaling pathways that regulate actin-filament uncapping and polymerization, in determining the role of myosin motor proteins in retrograde flow, and in evaluating the role of severing proteins in actin depolymerization. Both Listeria monocytogenes and Saccharomyces cerevisiae have emerged as powerful model organisms for studying actin dynamics in cells.

Actins↗
PubMed1997

Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding.

We have studied two types of cell motility directed toward the cell center: retraction of the cell margin and rearward flow of small cytoplasmic nodules during mitotic cell rounding in Potoroo tridactylis kidney (PtK2) cells by time-lapse video microscopy, drug treatments, and photoactivation of fluorescence. Nodules flow rearward on thin, actin-rich fibers (retraction fibers) exposed as the cell margin retracts. Retraction of the cell margin and rearward flow of nodules require intact actin filaments, but are insensitive to an inhibitor of myosin function (butanedione monoxime). Using photoactivation of fluorescence marking, we have determined that actin filaments in the majority of retraction fibers remain stationary while the cell margin retracts and nodules flow rearward. The pointed ends of retraction fiber actin filaments face the cell center. We argue that nodule motility is driven by a novel actin-based force that perhaps also partially contributes to retraction of the cell margin during cell rounding at mitosis.

Actins↗
PubMed1997

Microtubule polymerization dynamics.

The polymerization dynamics of microtubules are central to their biological functions. Polymerization dynamics allow microtubules to adopt spatial arrangements that can change rapidly in response to cellular needs and, in some cases, to perform mechanical work. Microtubules utilize the energy of GTP hydrolysis to fuel a unique polymerization mechanism termed dynamic instability. In this review, we first describe progress toward understanding the mechanism of dynamic instability of pure tubulin and then discuss the function and regulation of microtubule dynamic instability in living cells.

Animals↗
PubMed1996

Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.

Using a polymerization inhibition assay, we have purified a small, heat stable protein that physically interacts with tubulin dimers and increases the catastrophe rate of microtubules. Sequence analysis identified this protein as oncoprotein 18 (Op18)/stathmin, a conserved phosphoprotein that is highly expressed in leukemia cells. Immunodepletion experiments in Xenopus egg extracts showed that Op18/stathmin is involved in physiological regulation of mitotic microtubule dynamics. Op18/stathmin is a microtubule regulator that preferentially interacts with unpolymerized subunits. It is a candidate for increasing the microtubule catastrophe rate in mitosis and might also regulate microtubule dynamics in response to external signals.

Animals↗
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↗
PubMed1996

A purified Drosophila septin complex forms filaments and exhibits GTPase activity.

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.

Actin Cytoskeleton↗
PubMed1996

The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work.

During metaphase and anaphase in newt lung cells, tubulin subunits within the kinetochore microtubule (kMT) lattice flux slowly poleward as kMTs depolymerize at their minus-ends within in the pole. Very little is known about how and where the force that moves the tubulin subunits poleward is generated and what function it serves during mitosis. We found that treatment with the drug taxol (10 microM) caused separated centrosomes in metaphase newt lung cells to move toward one another with an average velocity of 0.89 microns/min, until the interpolar distance was reduced by 22-62%. This taxol-induced spindle shortening occurred as kMTs between the chromosomes and the poles shortened. Photoactivation of fluorescent marks on kMTs revealed that taxol inhibited kinetochore microtubule assembly/disassembly at kinetochores, whereas minus-end MT disassembly continued at a rate typical of poleward flux in untreated metaphase cells. This poleward flux was strong enough to stretch the centromeric chromatin between sister kinetochores as much as it is stretched in control metaphase cells. In anaphase, taxol blocked kMT disassembly/assembly at the kinetochore whereas minus-end disassembly continued at a rate similar to flux in control cells (approximately 0.2 microns/min). These results reveal that the mechanism for kMT poleward flux 1) is not dependent on kMT plus-end dynamics and 2) produces pulling forces capable of generating tension across the centromeres of bioriented chromosomes.

Anaphase↗
PubMed1995

Evolution of a dynamic cytoskeleton.

Actin filaments and microtubules form the cytoskeleton of all eukaryotic cells, and they are responsible for organizing the cytoplasm and supporting motile processes. Both polymers are highly dynamic, and their polymerization dynamics are central to their organization. Though their evolutionary origins appear to be distinct, actin and tubulin have a similar mechanism for promoting polymerization dynamics in which the energy of nucleotide triphosphate hydrolysis during polymerization is used to weaken the bonds between subunits, thus promoting subsequent depolymerization. The evolutionary origins of actin and tubulin are unclear. It is likely that motile mechanisms driven by reversible polymerization, termed thermal ratchets, are older than those based on ATPase motor proteins. Such mechanisms are still important in modern eukaryotes, and may have powered early versions of the critical motile processes of phagocytosis and chromosome segregation in primitive cells. Thus evolution of dynamic cytoskeletal polymers may have been one of the earliest and most important steps leading to the evolution of eukaryotes. Plausible evolutionary pathways can be constructed leading from simple enzymes to dynamic cytoskeletal polymers.

Actins↗
PubMed1995

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.

Amino Acid Sequence↗