Vertebrates vary in their ability to regenerate, and the genetic mechanisms underlying such disparity remain elusive. Comparative epigenomic profiling and single-cell sequencing of two related teleost fish uncovered species-specific and evolutionarily conserved genomic responses to regeneration. The conserved response revealed several regeneration-responsive enhancers (RREs), including an element upstream to inhibin beta A (inhba), a known effector of vertebrate regeneration. This element activated expression in regenerating transgenic fish, and its genomic deletion perturbed caudal fin regeneration and abrogated cardiac regeneration altogether. The enhancer is present in mammals, shares functionally essential activator protein 1 (AP-1)–binding motifs, and responds to injury, but it cannot rescue regeneration in fish. This work suggests that changes in AP-1–enriched RREs are likely a crucial source of loss of regenerative capacities in vertebrates.

Parthenogenetic species of whiptail lizards in the genus Aspidoscelis constitute a striking example of speciation by hybridization, in which first-generation hybrids instantly attain reproductive isolation and procreate as clonal all-female lineages. Production of eggs containing a full complement of chromosomes in the absence of fertilization involves genome duplication prior to the meiotic divisions. In these pseudo-tetraploid oocytes, pairing and recombination occur exclusively between identical chromosomes instead of homologs; a deviation from the normal meiotic program that maintains heterozygosity. Whether pseudo-tetraploid cells arise early in germ cell development or just prior to meiosis has remained unclear. We now show that in the obligate parthenogenetic species A. neomexicana the vast majority of oocytes enter meiosis as diploid cells. Telomere bouquet formation is normal, but synapsis fails and oocytes accumulate in large numbers at the pairing stage. Pseudo-tetraploid cells are exceedingly rare in early meiotic prophase, but they are the only cells that progress into diplotene. Despite the widespread failure to increase ploidy prior to entering meiosis, the fecundity of parthenogenetic A. neomexicana is similar to that of A. inornata, one of its bisexual ancestors.

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies.T HE organization, survival, and function of eukaryotic cells depend on intracellular transport governed by the microtubule-based molecular motors cytoplasmic dynein and kinesin. Dynein carries out the inward transport of cargos whereas kinesins are responsible for the outward movement. These motors, in addition to the transport and distribution of a wide variety of cargos, are also responsible for vital cellular processes ranging from mitosis to organelle positioning to embryonic development (Schroer et al. 1989;Burkhardt et al. 1997;Rana et al. 2004;Kardon and Vale 2009). Although intracellular transport is necessary for the function of all cells, polarized cells in particular have specific transport needs due to their asymmetry and elongated shape. These extraordinary requirements necessitate efficient longrange microtubule-based transport mechanisms (Hirokawa and Takemura 2005;Zheng et al. 2008;Harada 2010). The anterograde transport needs in these cells are satisfied by a variety of kinesins but only a single cytoplasmic dynein fulfills the retrograde transport requirements.Cytoplasmic dynein is a megadalton-sized, multiprotein complex composed of two heavy chains (DHCs), and varying numbers of dynein intermediate chains (DICs), light intermediate chains, and light chains. The DHCs perform the ATPase motor and microtubule-binding functions and the other subunits couple dynein to dynact...

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.T he vacuolar compartments in cells of fungi and plants have many of the same functions as the lysosomal compartments of animal cells (1-5). The defining characteristics of vacuoles and lysosomes are (i) the presence of a variety of hydrolytic enzymes such as proteases and phosphatases that help to degrade and recycle macromolecules and (ii) the maintenance of an acidic internal pH. An electrochemical gradient for protons is generated across vacuolar/lysosomal membranes by the vacuolar ATPase, a large complex enzyme (1, 6).We have been investigating the structure and function of the vacuolar compartments in filamentous fungi, using Neurospora crassa as our model organism. In fungi, vacuoles have multiple functions in addition to the degradation of macromolecules. They are involved in the regulation of arginine and ornithine metabolism, osmoregulation, and cytosolic ion and pH homeostasis. Fungal vacuoles contain high concentrations of basic amino acids, calcium, and polyphosphate (1, 4, 5).The vacuolar compartment in filamentous fungi has a complex structure and is highly dynamic (3,7,8). Many vacuoles have internal contents with a different refractive index than that of the cytosol, allowing them to be visualized with Nomarski optics. These vacuoles, prominent in older parts of the hypha, are spherical, ranging in size from Ͻ0.5 m to Ͼ10 m. Other vacuoles, prominent in the first hyphal segment, take the form of a dense network of interconnected tubules (9, 10). For N. cra...

Cytoplasmic dynein is responsible for the transport and delivery of cargoes in organisms ranging from humans to fungi. Dysfunction of dynein motor machinery due to mutations in dynein or its activating complex dynactin can result in one of several neurological diseases in mammals. The mouse Legs at odd angles (Loa) mutation in the tail domain of the dynein heavy chain has been shown to lead to progressive neurodegeneration in mice. The mechanism by which the Loa mutation affects dynein function is just beginning to be understood. In this work, we generated the dynein tail mutation observed in Loa mice into the Neurospora crassa genome and utilized cell biological and complementing biochemical approaches to characterize how that tail mutation affected dynein function. We determined that the Loa mutation exhibits several subtle defects upon dynein function in N. crassa that were not seen in mice, including alterations in dynein localization, impaired velocity of vesicle transport, and in the biochemical properties of purified motors. Our work provides new information on the role of the tail domain on dynein function and points out areas of future research that will be of interest to pursue in mammalian systems.