preparation-of-drosophila-s2-cells-for-light-microscopy

Preparation of <em>Drosophila</em> S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.

Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle.

Drosophila EB1 ist f&#xFC;r die ordnungsgem&#xE4;&#xDF;e Montage, Dynamik und Positionierung der mitotischen Spindel wichtig.

Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.

The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.

Die chromokinesin, KLP3A, Tauchg&#xE4;nge Mitosespindel Pol Trennung w&#xE4;hrend Prometaphase und Anaphase und erleichtert Chromatid Motilit&#xE4;t.

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.

Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase.

Zwei mitotischen Kinesinen zusammenarbeiten, um die Trennung der Schwesterchromatiden w&#xE4;hrend der Anaphase zu fahren.

Anaphase A chromatid-to-pole motion is fundamental for proper chromosome segregation in most systems. During the past several decades, two models for the mechanism of anaphase A have come to prominence. The Pacman model posits that chromatids induce the depolymerization of microtubule plus-ends embedded in kinetochores, thereby 'chewing' their way poleward. Alternatively, the Poleward-flux model posits that chromatids are 'reeled-in' to poles by the continual depolymerization of the minus-ends of kinetochore-associated microtubules, which are focused at spindle poles. In a recent study, we reported that anaphase A in Drosophila requires the depolymerization of both ends of kinetochore-associated microtubules, simultaneously. This is driven by two members of the Kin I subfamily of kinesins, termed KLP59C and KLP10A, which target specifically to chromatids and spindle poles, respectively. We have termed this hybrid of Pacman and Poleward flux the Kin I-dependent Pacman-flux mechanism for anaphase A. Here, we discuss the implications of these findings and explore potential additional components required to drive chromatid-to-pole motion by such a mechanism.

A Kin I-dependent Pacman-flux mechanism for anaphase A.

Ein Kin I-abh&#xE4;ngige Pacman-Flux-Mechanismus f&#xFC;r die Anaphase A.

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.

Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase.

Funktionell unterschiedlichen Kinesin-13 Familienmitglieder zusammen, um die Dynamik der Mikrotubuli w&#xE4;hrend der Interphase zu regulieren.

Accurate and timely chromosome segregation is a task performed within meiotic and mitotic cells by a specialized force-generating structure--the spindle. This micromachine is constructed from numerous proteins, most notably the filamentous microtubules that form a structural framework for the spindle and also transmit forces through it. Poleward flux is an evolutionarily conserved mechanism used by spindle microtubules both to move chromosomes and to regulate spindle length. Recent studies have identified a microtubule-depolymerizing kinesin as a key force-generating component required for flux. On the basis of these findings, we propose a new model for flux powered by a microtubule-disassembly mechanism positioned at the spindle pole. In addition, we use the flux model to explain the results of spindle manipulation experiments to illustrate the importance of flux for proper chromosome positioning.

Spindle microtubules in flux.

Spindel-Mikrotubuli im Fluss.

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150(glued), binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150(glued), we replaced the wild-type p150(glued) in Drosophila melanogaster S2 cells with mutant DeltaN-p150 lacking residues 1-200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150(glued) with DeltaN-p150(glued) has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150(glued) has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo.

Microtubule binding by dynactin is required for microtubule organization but not cargo transport.

Dctn3 verbindet cytoplasmatische Dynein und andere Motoren Cargo und engagiert sich in der Organisation von radial Microtubule-Arrays. Die gr&ouml;&szlig;te Untereinheit des DCTN3, p150(glued), bindet die Dynein-Mittelstufe-Kette und hat eine N-terminale Microtubule-bindende Dom&auml;ne. Um die Rolle des Microtubule-Bindung von p150(glued) zu untersuchen, ersetzt wir den Wildtyp-p150(glued) in Drosophila Melanogaster S2 Zellen mit Mutant Deltaner-p150 fehlt R&uuml;ckst&auml;nde 1-200, die Mikrotubuli binden kann. Zellen mit Cytochalasin D behandelt wurden f&uuml;r die Analyse von Ladung Bewegung entlang der Mikrotubuli verwendet. Auff&auml;llig ist, obwohl die Bewegung der membran&ouml;se Organellen und Kurier-Gebiets-komplexen durch Dynein und Kinesin-1 DCTN3 erfordert, hat die Substitution von in p150(glued) mit DeltaN-p150(glued) keine Auswirkung auf die Bewertung, Processivity oder Schrittgr&ouml;&szlig;e des Verkehrs. Allerdings hat das Abschneiden der Dom&auml;ne Microtubule-Bindung des p150(glued) einen dramatischen Effekt auf Zellteilung, was zu der Generation von multipolaren Spindeln und kostenlose Microtubule organizing Center. So ist DCTN3 Bindung an Mikrotubuli ben&ouml;tigt f&uuml;r die Organisation der Spindel Microtubule Arrays aber keine Ladung Motilit&auml;t in-vivo.

Microtubule-Bindung durch DCTN3 ist f&uuml;r Microtubule Organisation aber nicht lastentransport erforderlich.

Chromosomes move toward mitotic spindle poles by a Pacman-flux mechanism linked to microtubule depolymerization: chromosomes actively depolymerize attached microtubule plus ends (Pacman) while being reeled in to spindle poles by the continual poleward flow of tubulin subunits driven by minus-end depolymerization (flux). We report that Pacman-flux in Drosophila melanogaster incorporates the activities of three different microtubule severing enzymes, Spastin, Fidgetin, and Katanin. Spastin and Fidgetin are utilized to stimulate microtubule minus-end depolymerization and flux. Both proteins concentrate at centrosomes, where they catalyze the turnover of gamma-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing gamma-tubulin ring complexes from minus ends. In contrast, Katanin appears to function primarily on anaphase chromosomes, where it stimulates microtubule plus-end depolymerization and Pacman-based chromatid motility. Collectively, these findings reveal novel and significant roles for microtubule severing within the spindle and broaden our understanding of the molecular machinery used to move chromosomes.

Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes.

Chromosomen einen Schritt in Richtung mitotische Spindel Polen durch einen Pacman-Flux-Mechanismus verbunden Microtubule Depolymerisation: Chromosomen depolymerize aktiv angeschlossenen Microtubule zuz&uuml;glich enden (Pacman) zwar wieder zu Polen durch die kontinuierliche Mantelkante Str&ouml;mung Tubulin-Untereinheiten, die angetrieben von Minus-Ende Depolymerisation (Fluss)-Spindel, eingefangen. Wir berichten, dass Pacman-Flux in Drosophila Melanogaster die Aktivit&auml;ten der drei verschiedenen Microtubule ausgel&ouml;st Enzyme, Spastin, Fidgetin und Katanin umfasst. Spastin und Fidgetin werden eingesetzt, um Microtubule Minus-Ende Depolymerisation und Flussmittel zu stimulieren. Beide Proteine konzentrieren auf Centrosomes, wo sie katalysieren den Umsatz der Gamma-Tubulin, untermauern die Hypothese, dass sie ihren Einfluss nehmen, indem Sie freigeben, Gamma-Tubulin-Ring-komplexe aus abz&uuml;glich enden zu stabilisieren. Im Gegensatz dazu scheint Katanin Funktion in erster Linie bei Anaphase Chromosomen, wo es stimuliert Microtubule Plus-End Depolymerisation und Pacman-basierte Chromatid Motilit&auml;t. Gemeinsam diese Befunde zeigen, Roman und bedeutende Rollen f&uuml;r Microtubule Abbruch innerhalb der Spindel und erweitern unser Verst&auml;ndnis der molekularen Maschinerie verwendet, um Chromosomen zu verschieben.

Drei Microtubule ausgel&ouml;st Enzyme dazu beitragen, die &quot;Pacman-Flux&quot;-Maschinerie, die Chromosomen bewegt.

Cultured Drosophila cell lines have become an increasingly popular model system for cell biological and functional genomic studies. One of the most commonly used lines, S2 cells, is particularly useful as it is easy to grow and maintain in the lab, is highly susceptible to gene inhibition using RNAi and is well suited to high-resolution light microscopic assays. Here, we provide protocols for the routine culture and RNAi treatment of S2 cells and methods to prepare these cells for fluorescence microscopy. Using these techniques, loss-of-function experiments may be performed after 4-7 d of RNAi-mediated protein depletion.

Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy.

Kultivierte Drosophila-Zelllinien sind ein immer beliebter Modellsystem f&uuml;r Zelle biologischen und funktionale genomischer Studien geworden. Einer der wichtigsten gebr&auml;uchlichsten Linien, S2 Zellen, ist besonders n&uuml;tzlich, da es leicht zu wachsen und pflegen im Labor, ist sehr anf&auml;llig f&uuml;r gen-Hemmung mit RNAi und ist gut geeignet f&uuml;r hochaufl&ouml;sende Licht mikroskopischen Proben. Hier bieten wir Protokolle f&uuml;r die routinem&auml;&szlig;ige Kultur und RNAi Behandlung von S2-Zellen und Methoden, diese Zellen f&uuml;r die Fluoreszenzmikroskopie vorzubereiten. Mit diesen Techniken k&ouml;nnen Verlust der Funktion Experimente nach 4-7 d der RNAi-vermittelte Protein Ersch&ouml;pfung durchgef&uuml;hrt werden.

Kultur von Drosophila S2-Zellen und deren Verwendung f&uuml;r RNAi-vermittelte Verlustfunktion von Studien und Immunfluoreszenz-Mikroskopie.

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both gamma-tubulins. However, gamma-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted gamma-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.

A multicomponent assembly pathway contributes to the formation of acentrosomal microtubule arrays in interphase Drosophila cells.

In Tierzellen nucleate Centrosomes Mikrotubuli, die polarisierten Arrays Zytoplasma zu organisieren, zu bilden. Taufliege pr&auml;sentiert ein interessantes Paradox in tragf&auml;hige Erwachsene jedoch als Zentrosom-defizienten mutierte Tiere entwickeln. Um diese Diskrepanz zu verstehen, analysierten wir Verhalten von Centrosomes und Mikrotubuli in Drosophila Zellen, in der Kultur und in Vivo mit einer Kombination von Leben-Zelle Darstellung, Elektronenmikroskopie und RNAi. Das kanonische Modell des Zyklus der Zentrosom Funktion in Tierzellen Staaten Centrosomes wie Microtubule-Organisation handeln zentriert im gesamten Zellzyklus. Unerwartet, fanden wir, dass viele Drosophila-Zelltypen einen ver&auml;nderten Zyklus, Anzeigen in dem funktionalen Centrosomes nur w&auml;hrend der Zellteilung vorhanden sind. Auf mitotische Ausgang zerlegen Centrosomes produzierenden Interphase Zellen, die Centrioles, die Microtubule-Nukleierungsmittel Aktivit&auml;t fehlen. Dar&uuml;ber hinaus sind station&auml;re Zwischenphase Microtubule Ebenen nicht ver&auml;ndert codepleting beide Gamma-Tubulins. Jedoch verz&ouml;gert Gamma-Tubulin RNAi Microtubule nachwachsen nach Depolymerisation, was darauf hindeutet, dass es teilweise redundant funktionieren, mit einem anderen Weg. Daher wir zus&auml;tzliche Microtubule Nukleierungsmittel Faktoren untersucht und festgestellt, dass Mini-Spindeln, CLIP-190, ES1 oder Dynein RNAi auch verz&ouml;gert Microtubule nachwachsen; &Uuml;berraschenderweise wurde dies nicht weiter verl&auml;ngert wenn wir Gamma-Tubulins codepleted. Zusammen genommen, diese Ergebnisse &auml;ndern unsere Sicht des Zyklus der Zentrosom Funktion und offenbaren einen Mehrkomponenten Acentrosomal Microtubule Montage Weg Zwischenphase Microtubule-Arrays in Drosophila zu etablieren.

Multikomponenten Montage Weg tr&auml;gt zur Bildung von Acentrosomal Microtubule-Arrays in Interphase Drosophila Zellen.

Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

Einschr&auml;nken des Zentriol Vervielf&auml;ltigung auf einmal pro Zellzyklus ist entscheidend f&uuml;r Chromosome Segregation und genomischen Stabilit&auml;t, aber die Mechanismen dieser Block zu Reduplikation sind unklar. Genetische Untersuchungen haben eine Beteiligung f&uuml;r Skp/Cullin/F-Box (SCF) vorgeschlagen-Klasse Ubiquitin-Verkn&uuml;pfung in diesem Prozess. In dieser Studie beschreiben wir einen Mechanismus um Zentriol Reduplikation in Drosophila Melanogaster zu verhindern, wobei die SCF E3 Ubiquitin Ligase im Komplex mit dem F-Box-Protein Slimb proteolytischen Abbau der centrosomal regulatorische Kinase Plk4 vermittelt. Wir haben SCF(Slimb) als Regulator des Zentriol Vervielf&auml;ltigung &uuml;ber ein RNA-Interferenz (RNAi)-Bildschirm des Ubiquitin Cullin basierenden Verkn&uuml;pfung identifiziert. Wir fanden, dass Plk4 an Slimb bindet und ein SCF(Slimb) Ziel ist. Slimb und Plk4 zu Centrioles, mit Plk4 Ebenen h&ouml;chste bei Mitose zu lokalisieren und fehlt in S-Phase. Mit einem Plk4 Slimb-Bindung Mutant und Slimb RNAi, zeigen wir, dass Slimb Plk4 Lokalisierung zu Centrioles w&auml;hrend der Interphase, regelt somit Regulierung Zentriol Anzahl und die Gew&auml;hrleistung des Blocks Zentriol Reduplikation.

Der SCF Slimb Ubiquitin Ligase regelt Plk4/Sak Ebenen um Zentriol Reduplikation zu blockieren.

Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles - a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.

Centrosome function: sometimes less is more.

Enge Regelung der Zentrosom Vervielf&auml;ltigung ist entscheidend f&uuml;r das Zentrosom Anzahl Doppelzimmer nur einmal pro Zelle Zyklus zu gew&auml;hrleisten. &Uuml;berlagert dieser Zentrosom Vervielf&auml;ltigung-Zyklus ist ein funktionale Zentrosom-Zyklus, in dem sie zwischen Phasen der Ruhe und robuste Microtubule (MT) Keimbildung und MT-Verankerung Aktivit&auml;ten abwechseln. Interphase Centrioles sich in vertebrate Radfahren Zellen weniger Pericentriolar Material (PCM), ihre MT Keimbildung Kapazit&auml;tsabbau ansammeln. In Mitose Reifen Centrosomes, Anh&auml;ufung von mehr PCM um die Keimbildung zu erh&ouml;hen und Verankerung von Kapazit&auml;ten, robuste MT Astern zu bilden. Interessanterweise k&ouml;nnen funktionelle Zyklen der Centrosomes der Zelle Anforderungen entsprechend ge&auml;ndert werden. Einige Zwischenphase Centrosomes fungieren als Microtubule organizing Center durch die Erh&ouml;hung ihrer F&auml;higkeit, Anker MTs centrosomal radial Arrays zu bilden. Andere Zwischenphase Centrosomes pflegen ihre MT Keimbildung Kapazit&auml;t aber ihre MT-Verankerung-Kapazit&auml;t verringern/beseitigen. Aktuelle Arbeit zeigt, dass Drosophila Zellen nehmen dies auf die Spitze, wobei Centrioles verlieren alle nachweisbaren PCM w&auml;hrend der Interphase, bietet eine Erkl&auml;rung, wie Zentrosom-defizienten fliegen entwickeln bis zum Erwachsenenalter. Taufliege-Stammzellen weiter &auml;ndern den funktionalen Zyklus indem differentiell reguliert ihre zwei Centrioles - eine Situation, die wichtig f&uuml;r Stammzelle asymmetrische Teilungen, scheint wie Misregulation Zentrosom &Uuml;berschneidungen in Stammzellen und Vorl&auml;uferzellen Tumorbildung f&ouml;rdern kann. Hier &uuml;berpr&uuml;fen wir neuere Befunde, die Variationen in den funktionalen Kreislauf von Centrosomes zu beschreiben.

Zentrosom Funktion: weniger ist manchmal mehr.

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole-associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D's impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.

The Drosophila kinesin-13, KLP59D, impacts Pacman- and Flux-based chromosome movement.

Chromosom Bewegungen sind verbunden mit der aktiven Depolymerisation der Spindel Microtubule (MT) enden. Hier erkennen wir das Kinesin-13 Familienmitglied, KLP59D, als neuartige, eindeutig wichtiger Regulator der Spindel MT Dynamik und Chromosom Motilit&auml;t Drosophila somatischer Zellen. W&auml;hrend der Spindelapparat und Metaphase, Ersch&ouml;pfung der KLP59D, die Centrosomes und &auml;u&szlig;ere Kinetochores abzielt, unterdr&uuml;ckt die Depolymerisation der Spindel Pol-assoziierte MT abz&uuml;glich enden, was wiederum behindert Mantelkante Tubulin Flux. Sp&auml;ter, w&auml;hrend Anaphase, Verlust des KLP59D stark d&auml;mpft Chromatid-zu-Pol-Bewegung durch die Unterdr&uuml;ckung der Depolymerisation von beiden Minus und plus Enden der Kinetochor-assoziierte MTs. Der Mechanismus der KLP59Ds Auswirkungen auf Spindel MT plus und minus enden scheint zu unterscheiden. Unsere Daten unterst&uuml;tzen ein Modell in der KLP59D direkt w&auml;hrend der Anaphase depolymerizes Kinetochor-assoziierte zuz&uuml;glich enden, sondern Einfl&uuml;sse abz&uuml;glich enden indirekt durch Lokalisierung von die Pole Position-assoziierte MT depolymerase KLP10A. Schlie&szlig;lich gibt Elektronenmikroskopie, dass, wie 13 der anderen Drosophila Kinesin-s, KLP59D oligomerizing in MT-assoziierte Ringe in vitro weitgehend unf&auml;hig darauf hindeutet, dass solche Strukturen keine erforderliche Funktion Kinetochor-basierte MT Demontage und Chromosom Bewegungen sind.

Die Drosophila Kinesin-13, KLP59D, wirkt Pacman und Flux-basierte Chromosom Bewegung.

Regulated vascular endothelial growth factor (VEGF) signaling is required for proper angiogenesis, and excess VEGF signaling results in aberrantly formed vessels that do not function properly. Tumor endothelial cells have excess centrosomes and are aneuploid, properties that probably contribute to the morphologic and functional abnormalities of tumor vessels. We hypothesized that endothelial cell centrosome number is regulated by signaling via angiogenic factors, such as VEGF. We found that endothelial cells in developing vessels exposed to elevated VEGF signaling display centrosome overduplication. Signaling from VEGF, through either MEK/ERK or AKT to cyclin E/Cdk2, is amplified in association with centrosome overduplication, and blockade of relevant pathway components rescued the centrosome overduplication defect. Endothelial cells exposed to elevated FGF also had excess centrosomes, suggesting that multiple angiogenic factors regulate centrosome number. Endothelial cells with excess centrosomes survived and formed aberrant spindles at mitosis. Developing vessels exposed to elevated VEGF signaling also exhibited increased aneuploidy of endothelial cells, which is associated with cellular dysfunction. These results provide the first link between VEGF signaling and regulation of the centrosome duplication cycle, and suggest that endothelial cell centrosome overduplication contributes to aberrant angiogenesis in developing vessel networks exposed to excess angiogenic factors.

Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels.

Geregelten vaskul&auml;ren endothelialen Wachstumsfaktor (VEGF) Signalisierung ist erforderlich f&uuml;r die ordnungsgem&auml;&szlig;e Angiogenese und &uuml;bersch&uuml;ssige VEGF Signalisierung Ergebnisse in abnorm geformte Schiffe, die nicht gut funktionieren. Tumor Endothelzellen sind aneuploider und haben &uuml;bersch&uuml;ssige Centrosomes, Eigenschaften, wahrscheinlich auf die morphologischen und funktionellen Anomalien der Tumor Schiffe beitragen. Wir die Hypothese, dass Endothelzellen Zentrosom Zahl f&uuml;r die Signalisierung &uuml;ber angiogenen Faktoren wie VEGF geregelt ist. Wir fanden, dass Endothelzellen in der Entwicklung von Schiffen, erh&ouml;hte VEGF Signalisierung Display Zentrosom Overduplication ausgesetzt. Signalisierung von VEGF, entweder durch MEK/ERK oder AKT, cyclin E/Cdk2, wird verst&auml;rkt in Verbindung mit Zentrosom Overduplication und Blockade der entsprechenden Weg Komponenten rettete den Zentrosom Overduplication defekt. Endothelzellen, erh&ouml;hte FGF ausgesetzt hatte auch &uuml;bersch&uuml;ssige Centrosomes, was darauf hindeutet, dass mehrere angiogenen Faktoren Zentrosom Anzahl regulieren. Endothelzellen mit &uuml;bersch&uuml;ssigen Centrosomes &uuml;berlebte und aberrante Spindeln an Mitose gebildet. Entwicklung von Schiffen ausgesetzt erh&ouml;hten VEGF Signalisierung auch ausgestellt erh&ouml;hter Aneuploidie von Endothelzellen, zellulare Funktionsst&ouml;rung zugeordnet ist. Diese Ergebnisse stellen die erste Verbindung zwischen VEGF Signalisierung und Regulierung des Kreislaufs Vervielf&auml;ltigung Zentrosom und schlage vor, dass aberrante Angiogenesis in Entwicklung Schiff Netze, &uuml;bersch&uuml;ssige angiogenen Faktoren ausgesetzt, dass Endothelzellen Zentrosom Overduplication beitr&auml;gt.

Angiogenen Faktor Signalisierung regelt Zentrosom Vervielf&auml;ltigung in Endothelzellen der Blutgef&auml;&szlig;e zu entwickeln.

Do actin dynamics play an active role in mitotic spindle assembly? A new study demonstrates that cortical actin polymerization assists with the earliest phase of spindle pole migration.

Spindle assembly: more than just microtubules.

Spielen Aktin Dynamik eine aktive Rolle in mitotische Spindel Baugruppe? Eine neue Studie zeigt, dass die kortikale Aktin-Polymerisation mit der fr&uuml;hesten Phase der Spindel Pol Migration unterst&uuml;tzt.

Spindel Montage: mehr als nur Mikrotubuli.

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2A(Twins)) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification.

Zentriol Vervielf&auml;ltigung ist eine streng geregelte Prozess, der nur einmal pro Zellzyklus eintreten muss; Andernfalls k&ouml;nnen die &uuml;berz&auml;hligen Centrioles Aneuploidie und Tumorigenese ausl&ouml;sen. Plk4 (Polo-Like Kinase 4) Aktivit&auml;t initiiert Zentriol Vervielf&auml;ltigung und Ubiquitin vermittelten Proteolyse geregelt ist. In der gesamten Zwischenphase l&ouml;st Plk4 Autophosphorylierung seine Erniedrigung, und verhindert so Zentriol Verst&auml;rkung. Plk4 Aktivit&auml;t wird w&auml;hrend der Mitose zur richtigen Zentriol Duplizierung ben&ouml;tigt, jedoch der Stabilisierung mitotische Plk4 Mechanismus ist unbekannt. In diesem Beitrag zeigen wir, dass PP2A (Protein Phosphatase 2A(Twins)) wirkt Plk4 Autophosphorylierung, so Plk4 zu stabilisieren und Zentriol Vervielf&auml;ltigung zu f&ouml;rdern. Wie Plk4 die Protein-Ebene der Regulierung PP2As Untereinheit, Zwillinge (Tws), Spitzen w&auml;hrend der Mitose und f&uuml;r Zentriol Vervielf&auml;ltigung ist erforderlich. Jedoch stabilisiert vorzeitigen Tws-Ausdruck Plk4 unpassend, induzierende Zentriol Verst&auml;rkung. Paradoxerweise Ausdruck der Tumor-F&ouml;rderung simian Virus 40 kleine Tumor Antigen (ST), ein gemeldeten PP2A Hemmer, f&ouml;rdert Zentrosom Verst&auml;rkung durch einen unbekannten Mechanismus. Wir zeigen, dass ST eigentlich Tws-Funktion in Plk4 zu stabilisieren und induzierende Zentriol Verst&auml;rkung imitiert.

Article	Year
Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. The Journal of cell biology\| PubMed ID: 12213835	2002
The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility. Molecular biology of the cell\| PubMed ID: 14528012	2004
Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature\| PubMed ID: 14681690	2004
A Kin I-dependent Pacman-flux mechanism for anaphase A. Cell cycle (Georgetown, Tex.)\| PubMed ID: 15153812	2004
Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase. Nature cell biology\| PubMed ID: 15723056	2005
Spindle microtubules in flux. Journal of cell science\| PubMed ID: 15764594	2005
Microtubule binding by dynactin is required for microtubule organization but not cargo transport. The Journal of cell biology\| PubMed ID: 17325206	2007
Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes. The Journal of cell biology\| PubMed ID: 17452528	2007
Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy. Nature protocols\| PubMed ID: 18388942	2008
A multicomponent assembly pathway contributes to the formation of acentrosomal microtubule arrays in interphase Drosophila cells. Molecular biology of the cell\| PubMed ID: 18463166	2008
The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication. The Journal of cell biology\| PubMed ID: 19171756	2009
Centrosome function: sometimes less is more. Traffic (Copenhagen, Denmark)\| PubMed ID: 19192251	2009
The Drosophila kinesin-13, KLP59D, impacts Pacman- and Flux-based chromosome movement. Molecular biology of the cell\| PubMed ID: 19793918	2009
Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels. Blood\| PubMed ID: 20664058	2010
Spindle assembly: more than just microtubules. Current biology : CB\| PubMed ID: 21749958	2010
The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification. The Journal of cell biology\| PubMed ID: 21987638	2011

Gregory C. Rogers

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