Research Accomplishments of W.C. Earnshaw and Lab:

The research career of William C. Earnshaw has focused on the packaging and segregation of chromosomes in organisms ranging from bacte-riophages to mammalian cells. From the beginning of his Ph.D. research, in which he used a combination of ge-netics, biochemistry, electron microscopy, small-angle x-ray scattering and computer programme develop-ment, he has always emphasized a problem-oriented, rather than techniques-oriented approach. This approach has enabled him to make contributions in a number of areas, as detailed below.

brief synopsis of career: Postgraduate studies at MIT with Jonathan King led to a four year period of postdoctoral training at the MRC-LMB, under the sponsorship of Aaron Klug, where he worked closely with R.A. Crowther and Ron Laskey. A second one year postdoctoral stint with Ulrich Laemmli (Geneva) was followed by a move to the Johns Hopkins School of Medicine, where he stayed from 1981 to 1996, rising to the rank of Full Professor of Cell Biology and Associate Professor of Medicine. Since January 1996, Prof. Earnshaw has worked in the Institute of Cell and Molecular Biology at the University of Edinburgh as a Principal Research Fellow of The Wellcome Trust.

bacteriophage: Working with Stephen Harrison, he developed what is essentially the current model for the organization of packaged DNA. His thesis work in Jon King's lab at MIT established the paradigm of prohead expansion during virus assembly (collaboration with Sherwood Casjens).

mitotic chromosome structure: In collaboration with Leroy Liu, he and his student Margarete Heck were the first to show that DNA topoisomerase II is a major component of vertebrate chromosomes, located at the base of loop domains in the chromosome scaffold. Margarete then showed that the protein is expressed at high levels only in proliferating cells and that it undergoes differential phosphorylation across the cell cycle. His student Noriko Saitoh was the first to identify a condensin component, ScII/SMC2 as a major chromo-some scaffold component.

centromere/kinetochore structure and autoimmunity: He was the first to identify components of the human kinetochores: the human centromeric autoantigens (the CENP family = centromere proteins) using sera from scleroderma patients (a collaboration with rheumatologist, Naomi Rothfield). His student Becky Bernat then showed that the CENP antigens are required for assembly of functional kineto-chores. With Kevin Sullivan, he cloned CENP-B (80 kDa) and with Carol Cooke showed it to be distributed throughout the centromeric hete-rochromatin at ac-tive and inactive centromeres. Postdoctoral fellows John Tomkiel and Hisato Saitoh cloned CENP-C (140 kDa) and with Carol Cooke showed it to be a component of the DNA-rich in-ner kinetochore plate that is essential for de-termination of kinetochore size. Another postdoc, Peter Warburton, showed that CENP-A histone is concentrated in the inner kinetochore plate at active centromeres, providing the first evidence for a nucleosomal substructure of the vertebrate kinetochore. In other studies, his group identified the major scleroderma-associated autoantigen Scl-70 as DNA topoisomerase I and postdoc Peter D'Arpa was the first to clone human topo I. He developed ELISA assays for the CENP antigens, leading to the patenting of CENP-B and Topo I for immunodiagnostics. His student Bill Saunders was the first to clone human heterochro-matin protein HP1^Hsa. Antibodies raised against HP1^Hsa identified the PIKA, which is now emerging as a major subnuclear focus of proteins involved in sensing and repairing DNA damage.

chromosomal proteins and mitotic events: Working with Carol Cooke, he discovered INCENP, the inner centromere protein and used observations of its behaviour as the basis for the chromosome passenger hypothesis, which proposed that the chromosomes and cytoskeleton collaborate in the construction and operation of the molecular machinery that partitions daughter cells in mitosis. Postdoctoral fellows Richard Adams and Sally Wheatley have since identified Aurora-B kinase and Survivin/BIR-1 as chromosomal passenger proteins and shown that the three proteins interact with one another. INCENP is required for the correct targeting of Aurora-B and Survivin in mitosis. From the work of his group, the chromosomal passengers are emerging as major mitotic regulators that are essential for many mitotic functions, including kinetochore function and cytokinesis. These proteins are of clinical interest as all three are highly overexpressed in cancer cell lines. The most recent studies of his group include characterization of a conditional lethal INCENP mutant cell line (postdocs Paola Vagnarelli and Damien Hudson), characterization of drugs that affect chromosomal passenger function, and characterization of a link between chromosomal passengers and sister chromatid cohesion.

Apoptosis: Yuri Lazebnik, a postdoctoral fellow in his group, developed the first cell-free system reproducing the morpho-logi-cal and biochemical events of apoptotic execution, and in collaboration with Scott Kaufmann and Guy Poirier identified the first caspase substrate in apoptosis, poly(ADP-ribose) polymerase (PARP) and mapped its cleavage site (DEVD'G). Postdoctoral fellow Atsushi Takahashi identified the lamin A protease as caspase-6, and with Scott Kaufmann mapped the lamin A cleavage site as VEID'N. His group pioneered the use of affinity labeling of active caspases in apoptotic cells. Miguel Martins, a student, showed that certain active caspases are phosphoproteins. Another student, Kumiko Samejima, found that the transition from the latent to the execution phase of apoptosis marks a switch from a requirement for caspase function to the activation of downstream activities that drive nuclear disassembly in the absence of caspases. She, together with postdoctoral fellow Françoise Durrieu and Stefanie Kandels-Lewis identified two of these downstream activities as the caspase-activated DNase CAD and DNA topoisomerase II. Kumiko showed that, contrary to general belief, CAD and its regulatory chaperone ICAD/DFF45 are nuclear proteins. Most recently Kumiko, Sandrine Ruchaud and Nadia Korfali have prepared genetic knockouts of CAD and caspase-6 and are completing the characterization of the role of these factors in apoptotic execution.

Other activities: A major text book, Cell Biology, by T.D. Pollard and W.C. Earnshaw, with Graham Johnson (Publishers: Harcourt Health Sciences) is expected to be released late in 2001 or early in 2002. Prof. Earnshaw is a member of numerous editorial boards and serves on the Basic Science Interest Group of The Wellcome Trust.