Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti–Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes 1-4 , but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages 5 . Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expressionassociated epigenomic mark in eukaryotes.

The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction. INTRODUCTIONCytokinesis is the final step of cell division, required for the equal partitioning of the newly separated genetic material into two sister cells. Conventional animal cell cytokinesis is characterized by the assembly of an actin-myosin-based structure called the contractile ring, positioned midway between the two spindle poles, which drives the formation of a cleavage furrow. Despite its importance, many aspects of cytokinesis remain obscure. For example, although it is now well established that the cleavage plane can be specified by the central spindle or by astral microtubules (reviewed in Glotzer, 2001;Straight and Field, 2000), it is not known how microtubules trigger the assembly of the contractile ring or if they are required for regulating the contractile force.During the early phase of Drosophila embryogenesis, the embryo undergoes 13 rounds of nuclear division in the absence of cytokinesis. However, two kinds of membrane invagination events believed to be mechanistically related to conventional cytokinesis take place: metaphase furrow formation and cellularization. Metaphase furrows, or pseudocleavage furrows, form transiently during the 10th to 13th mitotic cycles of the cortical syncytial divisions. At interphase, actin is concentrated in cortical caps above each nucleus, but with entry into mitosis, the actin cytoskeleton reorganizes from caps to rings at the base of shallow furrows surrounding each developing spindle (Zalokar and Erk, 1976;Foe and Alberts, 1983;Karr and Alberts, 1986;Foe et al., 2000). These furrows fo...

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.

This paper asks how undergraduate engineering education supports students' ethical development, broadly defined, in a diverse sample of U.S. engineering schools and offers an analysis of the strengths and weaknesses of those efforts. The paper draws on observational case studies that were based on site visits to undergraduate mechanical and electrical engineering programs at seven universities or engineering schools in the U.S. It begins by proposing professional codes of ethics in engineering as a useful framework for thinking about the goals for student learning in the area of ethics and professional responsibility. The paper then discusses how and to what degree these goals are being addressed in the case study schools, with additional context provided through reference to published research in the field. The paper concludes with recommendations for strengthening the teaching of engineering ethics and professional responsibility.