TY - JOUR A1 - Tikhonenko, Irina A1 - Magidson, Valentin A1 - Gräf, Ralph A1 - Khodjakov, Alexey A1 - Koonce, Michael P. T1 - A kinesin-mediated mechanism that couples centrosomes to nuclei JF - Cellular and molecular life sciences N2 - The M-type kinesin isoform, Kif9, has recently been implicated in maintaining a physical connection between the centrosome and nucleus in Dictyostelium discoideum. However, the mechanism by which Kif9 functions to link these two organelles remains obscure. Here we demonstrate that the Kif9 protein is localized to the nuclear envelope and is concentrated in the region underlying the centrosome point of attachment. Nuclear anchorage appears mediated through a specialized transmembrane domain located in the carboxyl terminus. Kif9 interacts with microtubules in in vitro binding assays and effects an endwise depolymerization of the polymer. These results suggest a model whereby Kif9 is anchored to the nucleus and generates a pulling force that reels the centrosome up against the nucleus. This is a novel activity for a kinesin motor, one important for progression of cells into mitosis and to ensure centrosome-nuclear parity in a multinuclear environment. KW - Centrosome KW - Kinesin KW - Microtubule KW - Dictyostelium Y1 - 2013 U6 - https://doi.org/10.1007/s00018-012-1205-0 SN - 1420-682X VL - 70 IS - 7 SP - 1285 EP - 1296 PB - Springer CY - Basel ER - TY - JOUR A1 - Samereier, Matthias A1 - Baumann, Otto A1 - Meyer, Irene A1 - Gräf, Ralph T1 - Analysis of dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of dictyostelium microtubules JF - Cellular and molecular life sciences N2 - We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-alpha-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers. KW - Dictyostelium KW - TACC KW - DdCP224 KW - XMAP215 KW - Microtubules KW - Centrosome Y1 - 2011 U6 - https://doi.org/10.1007/s00018-010-0453-0 SN - 1420-682X VL - 68 IS - 2 SP - 275 EP - 287 PB - Springer CY - Basel ER - TY - THES A1 - Grafe, Marianne Erika T1 - Analysis of supramolecular assemblies of NE81, the first lamin protein in a non-metazoan organism T1 - Analyse von supramolekularen Komplexen von NE81, dem ersten Lamin in einem nicht-metazoischen Organismus N2 - Lamine sind Proteine an der inneren Kernhülle und bilden zusammen mit verbundenen Proteinen die nukleäre Lamina. Dieses Netzwerk sorgt für die Stabilität des Zellkerns und unterstützt die Organisation des Zell-Zytoskeletts. Zusätzlich sind Lamine und ihre verbundenen Proteine in viele Prozesse wie Genregulation und Zelldifferenzierung involviert. Bis 2012 war der Stand der Forschung, dass nur bei mehrzelligen Organismen eine nukleäre Lamina zu finden ist. NE81 ist das erste lamin-ähnliche Protein, das in einem nicht-mehrzelligen Organismus (Dictyostelium discoideum) entdeckt wurde. Es hat viele Eigenschaften und Strukturmerkmale mit Laminen gemeinsam. Dazu zählt der dreiteilige Aufbau des Proteins, eine Phosphorylierungsstelle für ein Zellzyklus-abhängiges Enzym, ein Kernlokalisationssignal, wodurch das Protein in den Kern transportiert wird, sowie eine C-terminale Sequenz zur Verankerung des Proteins in der Kernhülle. In dieser Arbeit wurden verschiedene Methoden zur vereinfachten Untersuchung von Laminstrukturen getestet, um zu zeigen, dass sich NE81 wie bereits bekannte Lamin-Proteine verhält und supramolekulare Netzwerke aus Laminfilamenten bildet. Zur Analyse der Struktur supramolekularer Anordnungen wurde das Protein durch Entfernen des Kernlokalisationssignals auf der äußeren Kernhülle von Dictyostelium gebildet. Die anschließende Untersuchung der Oberfläche der Kerne mit einem Rasterelektronenmikroskop zeigte, dass NE81 Strukturen in der Größe von Laminen bildet, allerdings nicht in regelmäßigen filamentösen Anordnungen. Um die Entstehung der Laminfilamente zu untersuchen, wurde lösliches NE81 aus Dictyostelium aufgereinigt und mit verschiedenen mikroskopischen Methoden untersucht. Dabei wurde festgestellt, dass NE81 unter Niedrigsalz-Bedingungen dünne, fadenförmige Strukturen und Netzwerke ausbildet, die denen von Säugetier-Laminen sehr ähnlich sind. Die Mutation der Phosphorylierungsstelle von NE81 zu einer imitierenden dauerhaften Phosphorylierung von NE81 in der Zelle, zeigte zunächst ein gelöstes Protein, das überraschenderweise unter Blaulichtbestrahlung der Zelle wieder lamin-ähnliche Anordnungen formte. Die Ergebnisse dieser Arbeit zeigen, dass NE81 echte Laminstrukturen ausbilden kann und hebt Dictyostelium als Nicht-Säugetier-Modellorganismus mit einer gut charakterisierten Kernhülle, mit allen relevanten, aus tierischen Zellen bekannten Proteinen, hervor. N2 - Nuclear lamins are nucleus-specific intermediate filaments forming a network located at the inner nuclear membrane of the nuclear envelope. They form the nuclear lamina together with proteins of the inner nuclear membrane regulating nuclear shape and gene expression, among others. The amoebozoan Dictyostelium NE81 protein is a suitable candidate for an evolutionary conserved lamin protein in this non-metazoan organism. It shares the domain organization of metazoan lamins and is fulfilling major lamin functions in Dictyostelium. Moreover, field-emission scanning electron microscopy (feSEM) images of NE81 expressed on Xenopus oocytes nuclei revealed filamentous structures with an overall appearance highly reminiscent to that of metazoan Xenopus lamin B2. For the classification as a lamin-like or a bona fide lamin protein, a better understanding of the supramolecular NE81 structure was necessary. Yet, NE81 carrying a large N-terminal GFP-tag turned out as unsuitable source for protein isolation and characterization; GFP-NE81 expressed in Dictyostelium NE81 knock-out cells exhibited an abnormal distribution, which is an indicator for an inaccurate assembly of GFP-tagged NE81. Hence, a shorter 8×HisMyc construct was the tag of choice to investi-gate formation and structure of NE81 assemblies. One strategy was the structural analysis of NE81 in situ at the outer nuclear membrane in Dictyostelium cells; NE81 without a func-tional nuclear localization signal (NLS) forms assemblies at the outer face of the nucleus. Ultrastructural feSEM pictures of NE81ΔNLS nuclei showed a few filaments of the expected size but no repetitive filamentous structures. The former strategy should also be established for metazoan lamins in order to facilitate their structural analysis. However, heterologously expressed Xenopus and C. elegans lamins showed no uniform localization at the outer nucle-ar envelope of Dictyostelium and hence, no further ultrastructural analysis was undertaken. For in vitro assembly experiments a Dictyostelium mutant was generated, expressing NE81 without the NLS and the membrane-anchoring isoprenylation site (HisMyc-NE81ΔNLSΔCLIM). The cytosolic NE81 clusters were soluble at high ionic strength and were purified from Dictyostelium extracts using Ni-NTA Agarose. Widefield immunofluorescence microscopy, super-resolution light microscopy and electron microscopy images of purified NE81 showed its capability to form filamentous structures at low ionic strength, as described previously for metazoan lamins. Introduction of a phosphomimetic point mutation (S122E) into the CDK1-consensus sequence of NE81 led to disassembled NE81 protein in vivo, which could be reversibly stimulated to form supramolecular assemblies by blue light exposure. The results of this work reveal that NE81 has to be considered a bona fide lamin, since it is able to form filamentous assemblies. Furthermore, they highlight Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope containing all rele-vant protein components known in animal cells. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina KW - expansion microscopy KW - Lamin KW - NE81 KW - Dictyostelium KW - Kernhülle KW - nukleäre Lamina KW - Expansions-Mikroskopie Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441802 ER - TY - JOUR A1 - Pitzen, Valentin A1 - Askarzada, Sophie A1 - Gräf, Ralph A1 - Meyer, Irene T1 - CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome JF - Cells N2 - Dictyostelium centrosomes consist of a nucleus-associated cylindrical, three-layered core structure surrounded by a corona consisting of microtubule-nucleation complexes embedded in a scaffold of large coiled-coil proteins. One of them is the conserved CDK5RAP2 protein. Here we focus on the role of Dictyostelium CDK5RAP2 for maintenance of centrosome integrity, its interaction partners and its dynamic behavior during interphase and mitosis. GFP-CDK5RAP2 is present at the centrosome during the entire cell cycle except from a short period during prophase, correlating with the normal dissociation of the corona at this stage. RNAi depletion of CDK5RAP2 results in complete disorganization of centrosomes and microtubules suggesting that CDK5RAP2 is required for organization of the corona and its association to the core structure. This is in line with the observation that overexpressed GFP-CDK5RAP2 elicited supernumerary cytosolic MTOCs. The phenotype of CDK5RAP2 depletion was very reminiscent of that observed upon depletion of CP148, another scaffolding protein of the corona. BioID interaction assays revealed an interaction of CDK5RAP2 not only with the corona markers CP148, gamma-tubulin, and CP248, but also with the core components Cep192, CP75, and CP91. Furthermore, protein localization studies in both depletion strains revealed that CP148 and CDK5RAP2 cooperate in corona organization. KW - centrosome KW - centriole KW - Dictyostelium KW - microtubules KW - mitosis Y1 - 2018 U6 - https://doi.org/10.3390/cells7040032 SN - 2073-4409 VL - 7 IS - 4 PB - MDPI CY - Basel ER - TY - JOUR A1 - Pitzen, Valentin A1 - Sander, Sophia A1 - Baumann, Otto A1 - Gräf, Ralph A1 - Meyer, Irene T1 - Cep192, a novel missing link between the centrosomal core and corona in Dictyostelium amoebae JF - Cells : open access journal N2 - The Dictyostelium centrosome is a nucleus-associated body with a diameter of approx. 500 nm. It contains no centrioles but consists of a cylindrical layered core structure surrounded by a microtubule-nucleating corona. At the onset of mitosis, the corona disassembles and the core structure duplicates through growth, splitting, and reorganization of the outer core layers. During the last decades our research group has characterized the majority of the 42 known centrosomal proteins. In this work we focus on the conserved, previously uncharacterized Cep192 protein. We use superresolution expansion microscopy (ExM) to show that Cep192 is a component of the outer core layers. Furthermore, ExM with centrosomal marker proteins nicely mirrored all ultrastructurally known centrosomal substructures. Furthermore, we improved the proximity-dependent biotin identification assay (BioID) by adapting the biotinylase BioID2 for expression in Dictyostelium and applying a knock-in strategy for the expression of BioID2-tagged centrosomal fusion proteins. Thus, we were able to identify various centrosomal Cep192 interaction partners, including CDK5RAP2, which was previously allocated to the inner corona structure, and several core components. Studies employing overexpression of GFP-Cep192 as well as depletion of endogenous Cep192 revealed that Cep192 is a key protein for the recruitment of corona components during centrosome biogenesis and is required to maintain a stable corona structure. KW - Cep192 KW - SPD-2 KW - centrosome KW - Dictyostelium KW - microtubule-organization KW - MTOC Y1 - 2021 U6 - https://doi.org/10.3390/cells10092384 SN - 2073-4409 VL - 10 IS - 9 PB - MDPI CY - Basel ER - TY - THES A1 - Kuhnert, Oliver T1 - Charakterisierung der neuen centrosomalen Proteine CP148 und CP55 in Dictyostelium discoideum T1 - Characterization of the new centrosomal proteins CP148 and CP55 in Dictyostelium discoideum N2 - Das im Cytosol liegende Dictyostelium Centrosom ist aus einer geschichteten Core-Region aufgebaut, die von einer Mikrotubuli-nukleierenden Corona umgeben ist. Zudem ist es über eine spezifische Verbindung eng an den Kern geknüpft und durch die Kernmembran hindurch mit den geclusterten Centromeren verbunden. Beim G2/M Übergang dissoziiert die Corona vom Centrosom und der Core verdoppelt sich so dass zwei Spindelpole entstehen. CP55 und CP148 wurden in einer Proteom-Analyse des Centrosoms identifiziert. CP148 ist ein neues coiled-coil Protein der centrosomalen Corona. Es zeigt eine zellzyklusabhängige An- und Abwesenheit am Centrosom, die mit der Dissoziation der Corona in der Prophase und ihrer Neubildung in der Telophase korreliert. Während der Telophase erschienen in GFP-CP148 exprimierenden Zellen viele, kleine GFP-CP148-Foci im Cytoplasma, die zum Teil miteinander fusionierten und zum Centrosom wanderten. Daraus resultierte eine hypertrophe Corona in Zellen mit starker GFP-CP148 Überexpression. Ein Knockdown von CP148 durch RNAi führte zu einem Verlust der Corona und einem ungeordneten Interphase Mikrotubuli-Cytoskelett. Die Bildung der mitotischen Spindel und der astralen Mikrotubuli blieb davon unbeeinflusst. Das bedeutet, dass die Mikrotubuli-Nukleationskomplexe während der Interphase und Mitose über verschiedene Wege mit dem Core assoziiert sind. Des Weiteren bewirkte der Knockdown eine Dispersion der Centromere sowie eine veränderte Sun1 Lokalisation in der Kernhülle. Somit spielt CP148 ebenso eine Rolle in der Centrosomen-Centromer-Verbindung. Zusammengefasst ist CP148 ein essentielles Protein für die Bildung und Organisation der Corona, welche wiederum für die Centrosom/Centromer Verbindung benötigt wird. CP55 wurde als Protein der Core-Region identifiziert und verbleibt während des Zellzyklus am Centrosom. Dort besitzt es strukturelle Aufgaben, da die Mehrheit der GFP-CP55 Moleküle in der Interphase keine Mobilität zeigten. Die GFP-CP55 Überexpression führte zur Bildung von überzähligen Centrosomen mit der üblichen Ausstattung an Markerproteinen der Corona und des Cores. CP55 Knockout-Zellen waren durch eine erhöhte Ploidie, eine weniger strukturierte und leicht vergrößerte Corona sowie zusätzliche cytosolische Mikrotubuli-organisierende Zentren charakterisiert. Letztere entstanden in der Telophase und enthielten nur Corona- aber keine Core-Proteine. In CP55 k/o Zellen erfolgte die Rekrutierung des Corona-Organisators CP148 an den Spindelpol bereits in der frühen Metaphase anstatt, wie üblich, erst in der Telophase. Außerdem zeigten die Knockout-Zellen Wachstumsdefekte, deren Grund vermutlich Schwierigkeiten bei der Centrosomenverdopplung in der Prophase durch das Fehlen von CP55 waren. Darüber hinaus konnten die Knockout-Zellen phagozytiertes Material nicht verwerten, obwohl der Vorgang der Phagozytose nicht beeinträchtigt war. Dieser Defekt kann dem im CP55 k/o auftretenden dispergierten Golgi-Apparat zugeschrieben werden. N2 - The Dictyostelium centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates yielding two spindle poles. The two proteins CP148 and CP55 were discovered in a proteomic analysis of Dictyostelium centrosomes. CP148 is a novel coiled-coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage. As CP148, CP55 was also identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed the majority of centrosomal GFP-CP55 is immobile indicating a structural task of CP55 at the centrosome. GFP-CP55 overexpression elicits supernumerary centrosomes containing the usual set of corona and core marker proteins. The CP55 null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their disorganized Golgi apparatus. KW - Dictyostelium KW - Centrosom KW - Mikrotubuli KW - Zellkern KW - Mitose KW - Dictyostelium KW - Centrosome KW - Microtubules KW - Nucleus KW - Mitosis Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-59949 ER - TY - JOUR A1 - Meyer, Irene A1 - Peter, Tatjana A1 - Batsios, Petros A1 - Kuhnert, Oliver A1 - Krueger-Genge, Anne A1 - Camurca, Carl A1 - Gräf, Ralph T1 - CP39, CP75 and CP91 are major structural components of the Dictyostelium JF - European journal of cell biology N2 - The acentriolar Dictyostelium centrosome is a nucleus-associated body consisting of a core structure with three plaque-like layers, which are surrounded by a microtubule-nucleating corona. The core duplicates once per cell cycle at the G2/M transition, whereby its central layer disappears and the two outer layers form the mitotic spindle poles. Through proteomic analysis of isolated centrosomes, we have identified CP39 and CP75, two essential components of the core structure. Both proteins can be assigned to the central core layer as their centrosomal presence is correlated to the disappearance and reappearance of the central core layer in the course of centrosome duplication. Both proteins contain domains with centrosome-binding activity in their N- and C-terminal halves, whereby the respective N-terminal half is required for cell cycle-dependent regulation. CP39 is capable of self-interaction and GFP-CP39 overexpression elicited supernumerary microtubule-organizing centers and pre-centrosomal cytosolic clusters. Underexpression stopped cell growth and reversed the MTOC amplification phenotype. In contrast, in case of CP75 underexpression of the protein by RNAi treatment elicited supernumerary MTOCs. In addition, CP75RNAi affects correct chromosome segregation and causes co-depletion of CP39 and CP91, another central core layer component. CP39 and CP75 interact with each other directly in a yeast two-hybrid assay. Furthermore, CP39, CP75 and CP91 mutually interact in a proximity-dependent biotin identification (BioID) assay. Our data indicate that these three proteins are all required for proper centrosome biogenesis and make up the major structural components of core structure's central layer. KW - Dictyostelium KW - Mitosis KW - Microtubules KW - Centrosome KW - Nucleus Y1 - 2017 U6 - https://doi.org/10.1016/j.eicb.2017.01.004 SN - 0171-9335 SN - 1618-1298 VL - 96 SP - 119 EP - 130 PB - Elsevier CY - Jena ER - TY - JOUR A1 - Kuhnert, Oliver A1 - Baumann, Otto A1 - Meyer, Irene A1 - Gräf, Ralph T1 - CP55, a novel key component of centrosomal organization in dictyostelium JF - Cellular and molecular life sciences N2 - Dictyostelium centrosomes consist of a layered core structure surrounded by a microtubule-nucleating corona. At the G2/M transition, the corona dissociates and the core structure duplicates, yielding two spindle pole bodies. Finally, in telophase, the spindle poles mature into two new, complete centrosomes. CP55 was identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed that during interphase the majority of centrosomal GFP-CP55 is immobile, which indicates a structural task of CP55 at the centrosome. The CP55null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their partially disorganized Golgi apparatus. KW - Dictyostelium KW - Corona KW - Microtubules KW - Centrosome KW - Nucleus Y1 - 2012 U6 - https://doi.org/10.1007/s00018-012-1040-3 SN - 1420-682X VL - 69 IS - 21 SP - 3651 EP - 3664 PB - Springer CY - Basel ER - TY - JOUR A1 - Putzler, Sascha A1 - Meyer, Irene A1 - Gräf, Ralph T1 - CP91 is a component of the Dictyostelium centrosome involved in centrosome biogenesis JF - European journal of cell biology N2 - The Dictyostelium centrosome is a model for acentriolar centrosomes and it consists of a three-layered core structure surrounded by a corona harboring microtubule nucleation complexes. Its core structure duplicates once per cell cycle at the G2/M transition. Through proteomic analysis of isolated centrosomes we have identified CP91, a 91-kDa coiled coil protein that was localized at the centrosomal core structure. While GFP-CP91 showed almost no mobility in FRAP experiments during interphase, both GFP-CP91 and endogenous CP91 dissociated during mitosis and were absent from spindle poles from late prophase to anaphase. Since this behavior correlates with the disappearance of the central layer upon centrosome duplication, CP91 is a putative component of this layer. When expressed as GFP-fusions, CP91 fragments corresponding to the central coiled coil domain and the preceding N-terminal part (GFP-CP91cc and GFP-CP91N, respectively) also localized to the centrosome but did not show the mitotic redistribution of the full length protein suggesting a regulatory role of the C-terminal domain. Expression of all GFP-fusion proteins suppressed expression of endogenous CP91 and elicited supernumerary centrosomes. This was also very prominent upon depletion of CP91 by RNAi. Additionally, CP91-RNAi cells exhibited heavily increased ploidy due to severe defects in chromosome segregation along with increased cell size and defects in the abscission process during cytokinesis. Our results indicate that CP91 is a central centrosomal core component required for centrosomal integrity, proper centrosome biogenesis and, independently, for abscission during cytokinesis. (c) 2016 Elsevier GmbH. All rights reserved. KW - Dictyostelium KW - Mitosis KW - Microtubules KW - Centrosome KW - Nucleus Y1 - 2016 U6 - https://doi.org/10.1016/j.ejcb.2016.03.001 SN - 0171-9335 SN - 1618-1298 VL - 95 SP - 124 EP - 135 PB - Royal Society CY - Jena ER - TY - JOUR A1 - Koonce, Michael A1 - Tikhonenko, Irina A1 - Gräf, Ralph T1 - Dictyostelium cell fixation BT - two simple tricks JF - Methods and protocols N2 - We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system. KW - Dictyostelium KW - cell fixation KW - microscopy KW - microtubule KW - cytoskeleton Y1 - 2020 U6 - https://doi.org/10.3390/mps3030047 SN - 2409-9279 VL - 3 IS - 3 PB - MDPI CY - Basel ER -