Reliable and sensitive analysis of occult bone marrow metastases using automated cellular imaging.

The presence of occult bone marrow metastases (OM) has been reported to represent an important prognostic indicator for patients with operable breast cancer and other malignancies. Assaying for OM most commonly involves labor-intensive manual microscopic analysis. The present report examines the performance of a recently developed automated cellular image analysis system (ACIS; ChromaVision Medical Systems, Inc.) for identifying and enumerating OM in human breast cancer specimens. OM analysis was performed after immunocytochemical staining. Specimens used in this study consisted of normal bone marrow (n = 10), bone marrow spiked with carcinoma cells (n = 20), and bone marrow obtained from breast cancer patients (n = 39). The reproducibility of ACIS-assisted analysis for tumor cell detection was examined by having a pathologist evaluate montage images generated from multiple ACIS runs of five specimens. Independent ACIS-assisted analysis resulted in the detection of an identical number of tumor cells for each specimen in all instrument runs. Additional studies were performed to analyze OM from 39 breast cancer patients with two pathologists performing parallel analysis using either manual microscopy or ACIS-assisted analysis. In 17 of the 39 cases (44%), specimens were classified by the pathologist as positive for tumor cells after ACIS-assisted analysis, whereas the same pathologist failed to identify tumor cells on the same slides after analysis by manual microscopy. These studies indicate that the ACIS-assisted analysis provides excellent sensitivity and reproducibility for OM detection, relative to manual microscopy. Such performance may enable an improved approach for disease staging and stratifying patients for therapeutic intervention.

Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

Cytoplasmic dynein function is essential in Drosophila melanogaster.

The microtubule motor cytoplasmic dynein has been implicated in a variety of intracellular transport processes. We previously identified and characterized the Drosophila gene Dhc64C, which encodes a cytoplasmic dynein heavy chain. To investigate the function of the cytoplasmic dynein motor, we initiated a mutational analysis of the Dhc64C dynein gene. A small deletion that removes the chromosomal region containing the heavy chain gene was used to isolate EMS-induced lethal mutations that define at least eight essential genes in the region. Germline transformation with a Dhc64C transgene rescued 16 mutant alleles in the single complementation group that identifies the dynein heavy chain gene. All 16 alleles were hemizygous lethal, which demonstrates that the cytoplasmic dynein heavy chain gene Dhc64C is essential for Drosophila development. Furthermore, our failure to recover somatic clones of cells homozygous for a Dhc64C mutation indicates that cytoplasmic dynein function is required for cell viability in several Drosophila tissues. The intragenic complementation of dynein alleles reveals multiple mutant phenotypes including male and/or female sterility, bristle defects, and defects in eye development.

Analyses of 22S dynein binding to Tetrahymena axonemes lacking outer dynein arms.

Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature of 39 degrees C. Axonemes isolated from nonmotile oad mutants (oad 39 degrees C axonemes) lack approximately 90% of their outer dynein arms and are deficient in 22S dynein. Here we report that oad 39 degrees C axonemes contain 40% of the 22S dynein heavy chains that wild-type axonemes contain and that oad axonemes do not undergo ATP-induced microtubule sliding in vitro. Wild-type 22S dynein will bind to the outer arm position in oad axonemes and restore ATP-induced microtubule sliding in those axonemes. Unlike wild-type 22S dynein, oad 22S dynein does not bind to the outer arm position in oad axonemes. These data indicate that the oad mutation affects some component of the outer arm dynein itself rather than the outer arm dynein binding site. These data also indicate that oad axonemes can be used to assay outer dynein arm function.

Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.

Biochemical analysis of a mutant Tetrahymena lacking outer dynein arms.

Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature, and axonemes isolated from nonmotile mutants lack approximately 90% of their outer dynein arms. Electrophoretic analyses of axonemes isolated from nonmotile mutants (oad axonemes) indicate they contain significantly fewer of the 22 S dynein heavy chains that axonemes isolated from wild-type cells (wild-type axonemes) contain. The 22 S dynein heavy chains that remain in axonemes isolated from nonmotile, oad mutants are assembled into 22 S dynein particles that exhibit wild-type levels of ATPase activity. Two-dimensional gel electrophoresis of oad axonemes show that they are deficient in no proteins other than those proteins thought to be components of 22 S dynein. This report is the first formal proof that outer dynein arms in Tetrahymena cilia are composed of 22 S dynein.