Jeffrey G. Ault

Centrosome and kinetochore movement during mitosis

During the past year important progress has been made in refining our understanding of how chromosomes become equally distributed to daughter cells during mitosis. Unlike the situation in diatoms and yeast, it now appears that spindle pole (centrosome) separation during spindle formation and anaphase B is mediated in vertebrates primarily by an astral pulling, and not a pushing, mechanism. Kinetochore motility is directionally unstable, which has important consequences for how chromosomes move to the equator of the forming spindle. Finally, the observation that sister chromatid disjunction occurs even in the presence of high levels of maturation promoting factor reveals that the series of biochemical events responsible for this phenomenon is not an obligatory part of the pathway by which the cell exits mitosis.

Adaptive changes in the kinetochore architecture facilitate proper spindle assembly

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that on mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore–microtubule interactions precedes the formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion–compaction in the context of lateral interactions correctly predict experimentally observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

Morphogenesis of the Mitotic and Meiotic Spindle: Conclusions Obtained From one System are not Necessarily Applicable to the Other

Chromosome distribution during both mitosis and meiosis is effected by the “spindle”, a complex ensemble formed from an interaction between chromosomes and microtubules (MTs). One of the most important characteristics of the spindle is its bipolar structure, established as it forms during prometaphase, which ensures that the replicated chromosomes are segregated equivalently to two daughter cells. A major goal of cell division research is to understand the mechanism of spindle morphogenesis and how bipolarity is established.

Palmitoyl-Protein Thioesterase 1 Deficiency in Drosophila melanogaster Causes Accumulation of Abnormal Storage Material and Reduced Life Span

Human neuronal ceroid lipofuscinoses (NCLs) are a group of genetic neurodegenerative diseases characterized by progressive death of neurons in the central nervous system (CNS) and accumulation of abnormal lysosomal storage material. Infantile NCL (INCL), the most severe form of NCL, is caused by mutations in the Ppt1 gene, which encodes the lysosomal enzyme palmitoyl-protein thioesterase 1 (Ppt1). We generated mutations in the Ppt1 ortholog of Drosophila melanogaster to characterize phenotypes caused by Ppt1 deficiency in flies. Ppt1-deficient flies accumulate abnormal autofluorescent storage material predominantly in the adult CNS and have a life span 30% shorter than wild type, phenotypes that generally recapitulate disease-associated phenotypes common to all forms of NCL. In contrast, some phenotypes of Ppt1-deficient flies differed from those observed in human INCL. Storage material in flies appeared as highly laminar spherical deposits in cells of the brain and as curvilinear profiles in cells of the thoracic ganglion. This contrasts with the granular deposits characteristic of human INCL. In addition, the reduced life span of Ppt1-deficient flies is not caused by progressive death of CNS neurons. No changes in brain morphology or increases in apoptotic cell death of CNS neurons were detected in Ppt1-deficient flies, even at advanced ages. Thus, Ppt1-deficient flies accumulate abnormal storage material and have a shortened life span without evidence of concomitant neurodegeneration.

The Drosophila protein palmitoylome: Characterizing palmitoyl- thioesterases and DHHC palmitoyl-transferases

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome’s normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.

Meiosis in Drosophila males

The conjunctive mechanism of the XY bivalent is believed to differ from that of the autosomal bivalents in the achiasmate Drosophila melanogaster male. It has been proposed that hypothetical cohesive elements, termed collochores, hold the X and Y chromosomes together at or near their nucleolar organizing regions (NORs) and that collochores are not exhibited by autosomal bivalents. In electron micrographs, unique fibrillar material is observed between the X and Y chromosomes at the synaptic site. Recently, the 240 bp nontranscribed spacer associated with rRNA genes at the NOR has been implicated as the essential DNA sequence for XY pairing. To test whether this DNA sequence is always associated with XY pairing and to determine its relationship to the unique fibrillar material, we studied the XY bivalent in Drosophila simulans. The D. simulans Y chromosome has few, if any, rRNA genes, but does have a large block (3,000 kb or 12,500 copies) of the nontranscribed spacer repeat located at the distal end of its long arm. This is in contrast to the D. melanogaster Y, which has the repeat located among rRNA genes on its short arm. Using light and electron microscopy, we show that the X does indeed pair with the distal end of the long arm of the D. simulans Y. However, no fibrillar material is evident in serial thin sections of the D. simulans XY bivalent, suggesting that this material (in D. melanogaster) may be remnants of the NOR rather than a morphological manifestation of the hypothetical collochores. Indeed, in electron micrographs, the synaptic regions of the XY and autosomal bivalents appear similar with no obvious pairing structures, suggesting that the conjunctive mechanism holding homologous chromosomes together is the same for the XY and autosomal bivalents.

Co-segregation of sex chromosomes in the male black widow spider Latrodectus mactans (Araneae, Theridiidae)

During meiosis I, homologous chromosomes join together to form bivalents. Through trial and error, bivalents achieve stable bipolar orientations (attachments) on the spindle that eventually allow the segregation of homologous chromosomes to opposite poles. Bipolar orientations are stable through tension generated by poleward forces to opposite poles. Unipolar orientations lack tension and are stereotypically not stable. The behavior of sex chromosomes during meiosis I in the male black widow spider Latrodectus mactans (Araneae, Theridiidae) challenges the principles governing such a scenario. We found that male L. mactans has two distinct X chromosomes, X1 and X2. The X chromosomes join together to form a connection that is present in prometaphase I but is lost during metaphase I, before the autosomes disjoin at anaphase I. We found that both X chromosomes form stable unipolar orientations to the same pole that assure their co-segregation at anaphase I. Using micromanipulation, immunofluorescence microscopy, and electron microscopy, we studied this unusual chromosome behavior to explain how it may fit the current dogma of chromosome distribution during cell division.

Investigating Kinetochore Structural Dynamics during Mitosis by Employing Combinations of Several Microscopic Techniques

The kinetochore attaches a chromosome to the mitotic spindle and harnesses forces that move the chromosome [1]. Recent studies indicate that kinetochores possess compliant linkages that contribute to mitotic checkpoint signaling [2]. Deformation of these elements are proposed to account for the differences in the distance between populations of labeled inner and outer plate proteins (intrakinetochore stretch) observed by light microscopy (LM). However, the underlying structural basis for intrakinetochore stretch remains unknown. To investigate this, we employ combinations of several microscopic techniques to investigate the change of the kinetochore from an expanded form to a contracted one during microtubule (MT) interaction, and how this change is reversed by drugs affecting MT polymerization. Fluorescent markers for outer and inner kinetochore proteins and for the spindle poles determine the orientation of sister kinetochores with respect to poles and to the centromere and allow precise determination of intrakinetochore stretch for individual kinetochores. By superimposing LM fluorescence on the distribution of gold particles in the corresponding EM images, we compare the shapes of kinetochores with high verses low values of intrakinetochore stretching (Figures 1 and 2).