W. Zacheus Cande: Evolutionary biologist in cell biologists clothing. Interview by Ruth Williams.

Zac Cande wants to get to the roots of cell division and cytoskeletal mechanics

A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells.

PtK1 cells lysed late in cell division in a medium containing the nonionic detergent Brij 58 and polyethylene glycol with continue to undergo cleavage after lysis. Maintenance of cleavage after lysis is dependent on the composition of the lysis medium; the pH must be around neutrality, MgATP must be present, and the free Ca++ concentration should be 1 microM for optimal constriction to occur. Cleavage can be stopped and reinitiated by raising and lowering the Ca++ levels in the lysis medium. Cleavage in the permeabilized cell is blocked by addition of phalloidin, cytochalasin B, and N-ethylmaleimide-modified myosin subfragment-1 to the lysis medium. This represents the first cell model system for studying cleavage since the pioneering studies of Hoffman-Berling in 1954

Chromosome movement in lysed mitotic cells is inhibited by vanadate.

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activty at less than millimolar concentrations. Vanadate at 10--100 micron reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate

Mad2, Bub3, and Mps1 regulate chromosome segregation and mitotic synchrony in Giardia intestinalis, a binucleate protist lacking an anaphase-promoting complex.

The binucleate pathogen Giardia intestinalis is a highly divergent eukaryote with a semiopen mitosis, lacking an anaphase-promoting complex/cyclosome (APC/C) and many of the mitotic checkpoint complex (MCC) proteins. However, Giardia has some MCC components (Bub3, Mad2, and Mps1) and proteins from the cohesin system (Smc1 and Smc3). Mad2 localizes to the cytoplasm, but Bub3 and Mps1 are either located on chromosomes or in the cytoplasm, depending on the cell cycle stage. Depletion of Bub3, Mad2, or Mps1 resulted in a lowered mitotic index, errors in chromosome segregation (including lagging chromosomes), and abnormalities in spindle morphology. During interphase, MCC knockdown cells have an abnormal number of nuclei, either one nucleus usually on the left-hand side of the cell or two nuclei with one mislocalized. These results suggest that the minimal set of MCC proteins in Giardia play a major role in regulating many aspects of mitosis, including chromosome segregation, coordination of mitosis between the two nuclei, and subsequent nuclear positioning. The critical importance of MCC proteins in an organism that lacks their canonical target, the APC/C, suggests a broader role for these proteins and hints at new pathways to be discovered

A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells.

After lysis in a Brij 58-polyethylene glycol medium, PtK1 cells are permeable to small molecules, such as erythrosin B, and to proteins, such as rhodamine-labeled FAB, myosin subfragment-1, and tubulin. Holes are present in the plasma membrane, and the mitochondria are swollen and distorted, but other membrane-bounded organelles of the lysed cell model are not noticeably altered. After lysis, the mitotic apparatus is functional; chromosomes move poleward and the spindle elongates. Cells lysed while in cytokinesis will continue to divide for several minutes. Addition of crude tubulin extracts, MAP-free tubulin, or taxol to the lysis medium retards anaphase chromosome movements but does not affect cleavage. On the other hand, N-ethylmaleimide-modified myosin subfragment-1, phalloidin, and cytochalasin B inhibit cleavage but have no effect on anaphase chromosome movements under identical lysis conditions. These results suggest that actomyosin plays no functional role in anaphase chromosome movement in mammalian tissue culture cells and that microtubule depolymerization is a rate-limiting step for chromosome-to-pole movements

The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation.

To study tubulin polymerization and microtubule sliding during spindle elongation in vitro, we developed a method of uncoupling the two processes. When isolated diatom spindles were incubated with biotinylated tubulin (biot-tb) without ATP, biot-tb was incorporated into two regions flanking the zone of microtubule overlap, but the spindles did not elongate. After biot-tb was removed, spindle elongation was initiated by addition of ATP. The incorporated biot-tb was found in the midzone between the original half-spindles. The extent and rate of elongation were increased by preincubation in biot-tb. Serial section reconstruction of spindles elongating in tubulin and ATP showed that the average length of half-spindle microtubules increased due to growth of microtubules from the ends of native microtubules. The characteristic packing pattern between antiparallel microtubules was retained even in the new overlap region. Our results suggest that the forces required for spindle elongation are generated by enzymes in the overlap zone that mediate the sliding apart of antiparallel microtubules, and that tubulin polymerization does not contribute to force generation. Changes in the extent of microtubule overlap during spindle elongation were affected by tubulin and ATP concentration in the incubation medium. Spindles continued to elongate even after the overlap zone was composed entirely of newly polymerized microtubules, suggesting that the enzyme responsible for microtubule translocation either is bound to a matrix in the spindle midzone, or else can move on one microtubule toward the spindle midzone and push another microtubule of opposite polarity toward the pole

In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts.

The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase

A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence

Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus; After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase

DSK1, a novel kinesin-related protein from the diatom Cylindrotheca fusiformis that is involved in anaphase spindle elongation.

We have identified an 80-kD protein that is involved in mitotic spindle elongation in the diatom Cylindrotheca fusiformis. DSK1 (Diatom Spindle Kinesin 1) was isolated using a peptide antibody raised against a conserved region in the motor domain of the kinesin superfamily. By sequence homology, DSK1 belongs to the central motor family of kinesin-related proteins. Immunoblots using an antibody raised against a non-conserved region of DSK1 show that DSK1 is greatly enriched in mitotic spindle preparations. Anti-DSK1 stains in diatom central spindle with a bias toward the midzone, and staining is retained in the spindle midzone during spindle elongation in vitro. Furthermore, preincubation with anti-DSK1 blocks function in an in vitro spindle elongation assay. This inhibition of spindle elongation can be rescued by preincubating concurrently with the fusion protein against which anti-DSK1 was raised. We conclude that DSK1 is involved in spindle elongation and is likely to be responsible for pushing hal-spindles apart in the spindle midzone

Telomeres act autonomously in maize to organize the meiotic bouquet from a semipolarized chromosome orientation

During meiosis, chromosomes undergo large-scale reorganization to allow pairing between homologues, which is necessary for recombination and segregation. In many organisms, pairing of homologous chromosomes is accompanied, and possibly facilitated, by the bouquet, the clustering of telomeres in a small region of the nuclear periphery. Taking advantage of the cytological accessibility of meiosis in maize, we have characterized the organization of centromeres and telomeres throughout meiotic prophase. Our results demonstrate that meiotic centromeres are polarized prior to the bouquet stage, but that this polarization does not contribute to bouquet formation. By examining telocentric and ring chromosomes, we have tested the cis-acting requirements for participation in the bouquet. We find that: (a) the healed ends of broken chromosomes, which contain telomere repeats, can enter the bouquet; (b) ring chromosomes enter the bouquet, indicating that terminal position on a chromosome is not necessary for telomere sequences to localize to the bouquet; and (c) beginning at zygotene, the behavior of telomeres is dominant over any centromere-mediated chromosome behavior. The results of this study indicate that specific chromosome regions are acted upon to determine the organization of meiotic chromosomes, enabling the bouquet to form despite large-scale changes in chromosome architecture