The Structure of the Mitotic Spindle and Nucleolus during Mitosis in the Amebo-Flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division

Distances and ages of globular clusters using Hipparcos parallaxes of local subdwarfs

We discuss the impact of Population II and Globular Cluster (GCs) stars on the derivation of the age of the Universe, and on the study of the formation and early evolution of galaxies, our own in particular. The long-standing problem of the actual distance scale to Population II stars and GCs is addressed, and a variety of different methods commonly used to derive distances to Population II stars are briefly reviewed. Emphasis is given to the discussion of distances and ages for GCs derived using Hipparcos parallaxes of local subdwarfs. Results obtained by different authors are slightly different, depending on different assumptions about metallicity scale, reddenings, and corrections for undetected binaries. These and other uncertainties present in the method are discussed. Finally, we outline progress expected in the near future.Comment: Invited review article to appear in: `Post-Hipparcos Cosmic Candles', A. Heck & F. Caputo (Eds), Kluwer Academic Publ., Dordrecht, in press. 22 pages including 3 tables and 2 postscript figures, uses Kluwer's crckapb.sty LaTeX style file, enclose

Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction

A Unique Role for Nonmuscle Myosin Heavy Chain IIA in Regulation of Epithelial Apical Junctions

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis

Immunohistochemical estimation of cell cycle phase in laryngeal neoplasia

We previously developed an immunohistochemical method for estimating cell cycle state and phase in tissue samples, including biopsies that are too small for flow cytometry. We have used our technique to examine whether primary abnormalities of the cell cycle exist in laryngeal neoplasia. Antibodies against the markers of cell cycle entry, minichromosome maintenance protein-2 (Mcm-2) and Ki67, and putative markers of cell cycle phase, cyclin D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis) were applied to paraffin-embedded sections of normal larynx (n=8), laryngeal dysplasia (n=10) and laryngeal squamous cell carcinoma (n=10). Cells expressing each marker were determined as a percentage of total cells, termed the labelling index (LI), and as a percentage of Mcm-2-positive cells, termed the labelling fraction (LF). The frequency of coexpression of each putative phase marker was investigated by confocal microscopy. There was a correlation between Mcm-2 and Ki67 LIs (ρ=0.93) but Mcm-2 LIs were consistently higher. All cells expressing a phase marker coexpressed Mcm-2, whereas Ki67 was not expressed in a proportion of these cells. The putative phase markers showed little coexpression. Labelling index values increased on progression from normal larynx through laryngeal dysplasia to squamous cell carcinoma for Mcm-2 (P=0.001), Ki67 (P=0.0002), cyclin D1 (P=0.015), cyclin A (P=0.0001) and cyclin B1 (P=0.0004). There was no evidence of an increase in the LF for any phase marker. Immunohistochemistry can be used to estimate cell cycle state and phase in laryngeal biopsies. Our data argues against primary cell cycle phase abnormalities in laryngeal neoplasia

Myosin II Motor Proteins with Different Functions Determine the Fate of Lamellipodia Extension during Cell Spreading

Non-muscle cells express multiple myosin-II motor proteins myosin IIA, myosin IIB and myosin IIC transcribed from different loci in the human genome. Due to a significant homology in their sequences, these ubiquitously expressed myosin II motor proteins are believed to have overlapping cellular functions, but the mechanistic details are not elucidated. The present study uncovered a mechanism that coordinates the distinctly localized myosin IIA and myosin IIB with unexpected opposite mechanical roles in maneuvering lamellipodia extension, a critical step in the initiation of cell invasion, spreading, and migration. Myosin IIB motor protein by localizing at the front drives lamellipodia extension during cell spreading. On the other hand, myosin IIA localizes next to myosin IIB and attenuates or retracts lamellipodia extension. Myosin IIA and IIB increase cell adhesion by regulating focal contacts formation in the spreading margins and central part of the spreading cell, respectively. Spreading cells expressing both myosin IIA and myosin IIB motor proteins display an organized actin network consisting of retrograde filaments, arcs and central filaments attached to focal contacts. This organized actin network especially arcs and focal contacts formation in the spreading margins were lost in myosin IIÂ cells. Surprisingly, myosin IIB̂ cells displayed long parallel actin filaments connected to focal contacts in the spreading margins. Thus, with different roles in the regulation of the actin network and focal contacts formation, both myosin IIA and IIB determine the fate of lamellipodia extension during cell spreading

Observational Constraints on the Common Envelope Phase

The common envelope phase was first proposed more than forty years ago to explain the origins of evolved, close binaries like cataclysmic variables. It is now believed that the phase plays a critical role in the formation of a wide variety of other phenomena ranging from type Ia supernovae through to binary black holes, while common envelope mergers are likely responsible for a range of enigmatic transients and supernova imposters. Yet, despite its clear importance, the common envelope phase is still rather poorly understood. Here, we outline some of the basic principles involved, the remaining questions as well as some of the recent observational hints from common envelope phenomena - namely planetary nebulae and luminous red novae - which may lead to answering these open questions.Comment: 29 pages, 8 figures. To appear in the book "Reviews in Frontiers of Modern Astrophysics: From Space Debris to Cosmology" (eds. Kabath, Jones and Skarka; publisher Springer Nature) funded by the European Union Erasmus+ Strategic Partnership grant "Per Aspera Ad Astra Simul" 2017-1-CZ01-KA203-03556

Shedding Light on the Galaxy Luminosity Function

From as early as the 1930s, astronomers have tried to quantify the statistical nature of the evolution and large-scale structure of galaxies by studying their luminosity distribution as a function of redshift - known as the galaxy luminosity function (LF). Accurately constructing the LF remains a popular and yet tricky pursuit in modern observational cosmology where the presence of observational selection effects due to e.g. detection thresholds in apparent magnitude, colour, surface brightness or some combination thereof can render any given galaxy survey incomplete and thus introduce bias into the LF. Over the last seventy years there have been numerous sophisticated statistical approaches devised to tackle these issues; all have advantages -- but not one is perfect. This review takes a broad historical look at the key statistical tools that have been developed over this period, discussing their relative merits and highlighting any significant extensions and modifications. In addition, the more generalised methods that have emerged within the last few years are examined. These methods propose a more rigorous statistical framework within which to determine the LF compared to some of the more traditional methods. I also look at how photometric redshift estimations are being incorporated into the LF methodology as well as considering the construction of bivariate LFs. Finally, I review the ongoing development of completeness estimators which test some of the fundamental assumptions going into LF estimators and can be powerful probes of any residual systematic effects inherent magnitude-redshift data.Comment: 95 pages, 23 figures, 3 tables. Now published in The Astronomy & Astrophysics Review. This version: bring in line with A&AR format requirements, also minor typo corrections made, additional citations and higher rez images adde

Relativistic Binaries in Globular Clusters

Galactic globular clusters are old, dense star systems typically containing 10\super{4}--10\super{7} stars. As an old population of stars, globular clusters contain many collapsed and degenerate objects. As a dense population of stars, globular clusters are the scene of many interesting close dynamical interactions between stars. These dynamical interactions can alter the evolution of individual stars and can produce tight binary systems containing one or two compact objects. In this review, we discuss theoretical models of globular cluster evolution and binary evolution, techniques for simulating this evolution that leads to relativistic binaries, and current and possible future observational evidence for this population. Our discussion of globular cluster evolution will focus on the processes that boost the production of hard binary systems and the subsequent interaction of these binaries that can alter the properties of both bodies and can lead to exotic objects. Direct {\it N}-body integrations and Fokker--Planck simulations of the evolution of globular clusters that incorporate tidal interactions and lead to predictions of relativistic binary populations are also discussed. We discuss the current observational evidence for cataclysmic variables, millisecond pulsars, and low-mass X-ray binaries as well as possible future detection of relativistic binaries with gravitational radiation.Comment: 88 pages, 13 figures. Submitted update of Living Reviews articl