Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va−) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.

Abstract. The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chsl) and two of them deficient in either Chs3 (calRI mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11͞Rad50͞Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.T he tumor suppressor gene BRCA1 was cloned several years ago through its link to inherited breast cancer (1). Since then, hundreds of mutations in the BRCA1 gene have been found in affected families. Approximately 50% of inherited breast cancer cases are estimated to result from BRCA1 mutations, and nearly all families with a history of both ovarian and breast cancer carry mutations in the gene (2). Studies of mammalian cells deficient in Brca1 have suggested that it is involved in DNA double-strand break repair, transcription-coupled repair, and cell cycle control, all of which are important for maintaining genomic stability (for a review, see ref.3).One of the early clues linking Brca1 to DNA repair was its association with Rad51, the primary RecA homolog in eukaryotic cells (4). The Brca1 protein colocalizes with Rad51 in nuclear dots during S phase and in response to DNA damage, suggesting that it may also be involved in homologous recombination and recombinational repair. The proliferation defects and embryonic lethality observed in mice with targeted disruptions of the BRCA1 gene (5-9) are very similar to the phenotypes of mice lacking Rad51 or Brca2, another factor associated with familial breast cancer (10, 11). All of these embryos are sensitive to ionizing radiation, exhibit high levels of chromosomal abnormalities, and can be partially rescued by p53 mutations.Recently, Brca1 was also found to associate with Rad50, part of the Mre11͞Rad50͞Nbs1(nibrin) complex (M͞R͞N) (12, 13), which is known to be involved in both nonhomologous endjoining and homologous recombination in yeast and vertebrate cells (14-18). The Nbs1 component of the complex is phosphorylated in response to DNA damage by ATM (19-22), a kinase that also phosphorylates Brca1 after the introduction of doublestrand breaks (23, 24). The Brca1 foci, which appear after ionizing radiation, colocalize, in a subset of the cell population, with nuclear foci formed by M͞R͞N (12, 13), again suggesting a link between Brca1 and the cellular response to DNA doublestrand breaks. How these foci form and what draws Brca1 to these foci are unknown.Another consequence of ionizing radiation is the accumulation of oxidized bases, which are removed preferentially from transcriptionally active genes in a pr...