Timothy J. Mitchison

Actin-Based Cell Motility and Cell Locomotion

We explicitly acknowledge key intellectual contributions from Jody Rosenblatt (depolymerization) and Julie Theriot (protrusion, Listeria motility). Owing to space restrictions, we were unable to cite all relevant publications. We attempted to fairly represent different laboratories, systems, and opinions and apologize to authors whose papers we did not include. We thank Matt Welch and Jody Rosenblatt for helpful comments on the manuscript. This work was funded by grant GM48027 from the National Institutes of Health and a fellowship from the Packard Foundation to T. J. Mitchison, and a senior postdoctoral fellowship from the American Cancer Society to L. P. Cramer.

A chemical method for fast and sensitive detection of DNA synthesis in vivo

We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (“click” chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.

A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish

Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H2O2), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H2O2 during the early events of wound responses in zebrafish larvae expressing a genetically encoded H2O2 sensor. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting ∼3 min after wounding and peaking at ∼20 min, which extended ∼100–200 μm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role.

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.

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.

Preparation of modified tubulins

Publisher Summary This chapter presents a collection of the various different ways by which tubulins are modified to generate probes for investigating microtubule (MT) dynamics in vitro and in vivo . Labeling with biotin and various fluorochromes is described, as well as the preparation of N-ethylmaleimide tubulin, which has been used to block minus-end growth in vitro . The use of GTP analogs to prepare stable labeled microtubules has proved very useful in a number of different experiments. The tubulin used in the presented methods was prepared from bovine brain by two cycles of temperature-dependent polymerization, followed by phosphocellulose chromatography. The cycling procedure described in the chapter selects active subunits and removes free nucleotide. This produces a tubulin preparation suitable for use in in vitro assays. The standard biotin-labeled tubulin preparation has been used to determine sites of microtubule elongation in vivo and in vitro . It is difficult to quantitate the stoichiometry of biotin labeling on a routine basis, but early work using radioactive N-hydroxysuccinimide (NHS)-biotin gave a labeling stochiometry of one to three biotins/tubulin dimer. The final yield of twice cycled biotin-tubulin is about 10% of the starting protein. Tetramethylrhodamine-labeled tubulin has been used to follow microtubules in living cells and it is also used for marking microtubules in real-time in vitro assays.

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.

Identification of small-molecule inhibitors of interaction between the BH3 domain and Bcl-xL

To study the role of the BH3 domain in mediating pro-apoptotic and anti-apoptotic activities of Bcl-2 family members, we identified a series of novel small molecules (BH3Is) that inhibit the binding of the Bak BH3 peptide to Bcl-xL. NMR analyses revealed that BH3Is target the BH3-binding pocket of Bcl-xL. Inhibitors specifically block the BH3-domain-mediated heterodimerization between Bcl-2 family members in vitro and in vivo and induce apoptosis. Our results indicate that BH3-dependent heterodimerization is the key function of anti-apoptotic Bcl-2 family members and is required for the maintenance of cellular homeostasis.

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.

A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro

We report here a chromosomal protein that plays an essential role in mitotic chromosome condensation in Xenopus egg extracts. Two polypeptides, designated XCAP-C and XCAP-E, were found to associate with each other in the extracts, presumably forming a heterodimer. During chromosome assembly in mitotic extracts, XCAP-C/E was recruited to the chromatin and formed a discrete internal structure within assembled chromosomes. Antibody blocking experiments showed that XCAP-C function is required for both assembly and structural maintenance of mitotic chromosomes in vitro. Deduced amino acid sequences revealed that the two polypeptides share common structural motifs, consisting of an N-terminal NTP-binding domain, two central coiled-coil regions, and a C-terminal conserved domain. These motifs are highly conserved in a protein family, members of which have been identified recently in both prokaryotes and eukaryotes.