Genetic assessments and parentage analysis of captive Bolson tortoises (Gopherus flavomarginatus) inform their "rewilding" in New Mexico.

The Bolson tortoise (Gopherus flavomarginatus) is the first species of extirpated megafauna to be repatriated into the United States. In September 2006, 30 individuals were translocated from Arizona to New Mexico with the long-term objective of restoring wild populations via captive propagation. We evaluated mtDNA sequences and allelic diversity among 11 microsatellite loci from the captive population and archived samples collected from wild individuals in Durango, Mexico (n = 28). Both populations exhibited very low genetic diversity and the captive population captured roughly 97.5% of the total wild diversity, making it a promising founder population. Genetic screening of other captive animals (n = 26) potentially suitable for reintroduction uncovered multiple hybrid G. flavomarginatus×G. polyphemus, which were ineligible for repatriation; only three of these individuals were verified as purebred G. flavomarginatus. We used these genetic data to inform mate pairing, reduce the potential for inbreeding and to monitor the maintenance of genetic diversity in the captive population. After six years of successful propagation, we analyzed the parentage of 241 hatchlings to assess the maintenance of genetic diversity. Not all adults contributed equally to successive generations. Most yearly cohorts of hatchlings failed to capture the diversity of the parental population. However, overlapping generations of tortoises helped to alleviate genetic loss because the entire six-year cohort of hatchlings contained the allelic diversity of the parental population. Polyandry and sperm storage occurred in the captives and future management strategies must consider such events.

Analysis of centrosome function and microtubule dynamics by time-lapse microscopy in Xenopus egg extracts.

Centrosomes are essential organelles that organize the microtubule cytoskeleton during interphase and mitosis. Centrosomes are assembled from tens to hundreds of proteins, but how these proteins are organized into functional microtubule nucleating and organizing centers is not yet clear. An important step in understanding the role of individual proteins in centrosome function is to understand whether they are involved in forming, stabilizing, or anchoring microtubules. It is becoming increasingly clear that the analysis of fixed samples is inadequate for a true understanding of the dynamics that drive cell biological processes. In this chapter we focus on methods to analyze microtubule nucleation, organization, and dynamics using assays based on mitotic Xenopus egg extracts and in vitro reactions. These methods can easily be adapted to the study of interphase processes, or to the study of other cytoskeletal proteins and their dynamics.

Myosin-10 and actin filaments are essential for mitotic spindle function.

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.

Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome.

Members of the transforming acidic coiled coil (TACC) protein family are emerging as important mitotic spindle assembly proteins in a variety of organisms. The molecular details of how TACC proteins function are unknown, but TACC proteins have been proposed to recruit microtubule-stabilizing proteins of the tumor overexpressed gene (TOG) family to the centrosome and to facilitate their loading onto newly emerging microtubules. Using Xenopus egg extracts and in vitro assays, we show that the Xenopus TACC protein maskin is required for centrosome function beyond recruiting the Xenopus TOG protein XMAP215. The conserved C-terminal TACC domain of maskin is both necessary and sufficient to restore centrosome function in maskin-depleted extracts, and we provide evidence that the N terminus of maskin inhibits the function of the TACC domain. Time-lapse video microscopy reveals that microtubule dynamics in Xenopus egg extracts are unaffected by maskin depletion. Our results provide direct experimental evidence of a role for maskin in centrosome function and suggest that maskin is required for microtubule anchoring at the centrosome.

Xenopus NEDD1 is required for microtubule organization in Xenopus egg extracts.

The centrosome serves as the major microtubule-nucleating and -organizing center in animal cells. It is composed of hundreds of proteins. The molecular details of how centrosomal proteins contribute to centrotome function are only beginning to emerge. Members of the neuron-precursor-cell-expressed developmentally downregulated protein 1 (NEDD1) family of conserved proteins have recently been implicated in recruiting gamma-tubulin and its associated proteins, which together make up the gamma-tubulin ring complex (gammaTuRC), to the centrosome. Human NEDD1 and its Drosophila ortholog Dgp71WD are WD-repeat proteins that interact with the gammaTuRC. Experimental knockdown of human NEDD1 was recently shown to result in loss of gamma-tubulin from the centrosome. By contrast, however, Dgp71WD knockdown has no effect on targeting the gammaTuRC to the centrosome in flies. Using Xenopus egg extracts, we show that Xenopus NEDD1 is mostly dispensable for targeting gamma-tubulin to centrosomes, but that microtubule organization is disrupted in NEDD1-depleted extracts. We show that NEDD1 exists in a complex that is distinct from the gammaTuRC, suggesting that NEDD1 may not be a bona fide subunit of the Xenopus gammaTuRC. We propose that the main function of NEDD1 in Xenopus is in microtubule organization.

Distinct Dgrip84 isoforms correlate with distinct gamma-tubulins in Drosophila.

Gamma-tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, gamma-tubulin exists in a multiprotein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). gamma-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the gamma-tubulin ring complex (gammaTuRC). Curiously, many organisms (including humans) have two distinct gamma-tubulin genes. In Drosophila, where the two gamma-tubulin isotypes have been studied most extensively, the gamma-tubulin genes are developmentally regulated: the "maternal" gamma-tubulin isotype (named gammaTub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second gamma-tubulin isotype (gammaTub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both gamma-tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes: gammaTub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of gammaTub23C at the centrosome increases at the beginning of mitosis, and gammaTub23C also associates with spindle pole microtubules. Here, we show that gammaTub23C forms discrete complexes that closely resemble the complexes formed by gammaTub37CD. Surprisingly, however, gammaTub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the gamma-tubulin complexes formed with gammaTub37CD and gammaTub23C.

Phosphorylation of maskin by Aurora-A is regulated by RanGTP and importin beta.

Mitotic spindle assembly in Xenopus egg extracts is regulated at least in part by importin beta and its regulator, the small GTPase, Ran. RanGTP stabilizes microtubules near the chromosomes during spindle assembly by selectively releasing spindle assembly factors from inhibition by importin alpha/beta in the vicinity of the chromosomes. Several spindle assembly factors are regulated in this manner. We identified maskin, the Xenopus member of the transforming acidic coiled coil family of proteins, as a potential candidate in a two-step affinity chromatography approach designed to uncover additional downstream targets of importin alpha/beta in mitosis. Here, we show that although maskin lacks a canonical nuclear localization sequence, it binds importin beta in a RanGTP-regulated manner. We further show that importin beta inhibits the regulatory phosphorylation of maskin by Aurora-A. This suggests a novel mechanism by which importin beta regulates the activity of a spindle assembly factor.

Microtubule nucleation: gamma-tubulin and beyond.

Centrosomes and their fungal equivalents, spindle pole bodies (SPBs), are the main microtubule (MT)-organizing centers in eukaryotic cells. Several proteins have been implicated in microtubule formation by centrosomes and SPBs, including microtubule-minus-end-binding proteins and proteins that bind along the length or stabilize the plus ends of microtubules. Recent work has improved our understanding of the molecular mechanisms of MT formation. In particular, it has shown that gamma-tubulin and its associated proteins play key roles in microtubule nucleation and spindle assembly in evolutionarily distant species ranging from fungi to mammals. Other work indicates that gamma-tubulin-mediated microtubule nucleation, although necessary, is not sufficient for mitotic spindle assembly but requires additional proteins that regulate microtubule nucleation independently of centrosomes.

What's so Bor(a)ing about Aurora-A activation?

Aurora-A kinases are highly conserved mitotic kinases required for cell division. The regulation of Aurora-A activity is less highly conserved and currently poorly understood. Work by Knoblich and coworkers in this issue of Developmental Cell identifies the conserved protein, Aurora Borealis (Bora), as a key regulator of Aurora-A activity during mitosis.

A picornavirus protein interacts with Ran-GTPase and disrupts nucleocytoplasmic transport.

Active nucleocytoplasmic transport of protein and RNA in eukaryotes depends on the Ran-GTPase system to regulate cargo-receptor interactions. Several viruses, including the RNA picornaviruses, encode factors that alter nuclear transport with the aim of suppressing synthesis of antiviral factors and promoting viral replication. Picornaviruses in the cardiovirus genus express a unique 67-aa Leader protein (L), known to alter the subcellular distribution of IFN regulatory proteins targeted to the nucleus. We report here that L binds directly to Ran and blocks nuclear export of new mRNAs. In Xenopus egg extracts, recombinant L also inhibits mitotic spindle assembly, a RanGTP function crucial to cell-cycle progression. We propose that L inhibits nucleocytoplasmic transport during infection by disrupting the RanGDP/GTP gradient. This inhibition triggers an efflux of nuclear proteins necessary for viral replication and causes IFN suppression. To our knowledge, L is the first viral picornaviral protein to interact directly with Ran and modulate the Ran-dependent nucleocytoplasmic pathway.

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts.

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.

The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly.

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a approximately 200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to "rescue" experiments. Defects in the "early" step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the "late" step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.

Nuclear and mitochondrial localization signals overlap within bovine herpesvirus 1 tegument protein VP22.

VP22, a tegument protein of bovine herpesvirus 1, accumulates in the nucleus of infected and transiently transfected cells. Previous studies indicated a possible regulatory function of VP22 within nuclei, but how VP22 enters nuclei is unknown. Despite the abundance of basic residues within this protein, no classic nuclear localization signal (NLS) motif has been identified. To identify the signal directing nuclear accumulation, a series of truncations, internal deletions, and point mutations were constructed. Fluorescence microscopy of cells transfected with VP22 constructs indicated that a sequence of 103 residues is necessary and sufficient for nuclear localization. This NLS sequence is conformation-sensitive in contrast to a classical sequential NLS. Energy depletion assays and co-immunoprecipitation suggested that this NLS sequence also binds histone H4, resulting in nuclear retention of VP22. In addition, a mitochondrial targeting sequence was identified at the C-terminal 49 amino acids, which overlapped the sequence required for nuclear targeting. Our findings demonstrate the diversity of VP22 protein to localize within the cell and provide the opportunity for VP22 to direct cargo specifically to different subcellular compartments.

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly.

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.