TY - THES A1 - Anielski, Alexander T1 - Entwicklung einer mikrofluidischen, adaptiv geregelten Messapparatur zur quantitativen Untersuchung von Chemotaxis mit Hilfe der Flussfotolyse KW - Flussfotolyse KW - Konzentration KW - Chemotaxis KW - Mikrokanal KW - Dictyostelium KW - flow photolysis KW - concentration KW - chemotaxis KW - microchannel KW - Dictyostelium Y1 - 2015 ER - TY - THES A1 - Barbosa Pfannes, Eva Katharina T1 - Probing the regulatory mechanisms of the actomyosin system in motile cells T1 - Erforschung von Regulationsmechanismen des Aktomyosinsystems in bewegliche Zellen N2 - Actin-based directional motility is important for embryonic development, wound healing, immune responses, and development of tissues. Actin and myosin are essential players in this process that can be subdivided into protrusion, adhesion, and traction. Protrusion is the forward movement of the membrane at the leading edge of the cell. Adhesion is required to enable movement along a substrate, and traction finally leads to the forward movement of the entire cell body, including its organelles. While actin polymerization is the main driving force in cell protrusions, myosin motors lead to the contraction of the cell body. The goal of this work was to study the regulatory mechanisms of the motile machinery by selecting a representative key player for each stage of the signaling process: the regulation of Arp2/3 activity by WASP (actin system), the role of cGMP in myosin II assembly (myosin system), and the influence of phosphoinositide signaling (upstream receptor pathway). The model organism chosen for this work was the social ameba Dictyostelium discoideum, due to the well-established knowledge of its cytoskeletal machinery, the easy handling, and the high motility of its vegetative and starvation developed cells. First, I focused on the dynamics of the actin cytoskeleton by modulating the activity of one of its key players, the Arp2/3 complex. This was achieved using the carbazole derivative Wiskostatin, an inhibitor of the Arp2/3 activator WASP. Cells treated with Wiskostatin adopted a round shape, with no of few pseudopodia. With the help of a microfluidic cell squeezer device, I could show that Wiskostatin treated cells display a reduced mechanical stability, comparable to cells treated with the actin disrupting agent Latrunculin A. Furthermore, the WASP inhibited cells adhere stronger to a surface and show a reduced motility and chemotactic performance. However, the overall F-actin content in the cells was not changed. Confocal microscopy and TIRF microscopy imaging showed that the cells maintained an intact actin cortex. Localized dynamic patches of increased actin polymerization were observed that, however, did not lead to membrane deformation. This indicated that the mechanisms of actin-driven force generation were impaired in Wiskostatin treated cells. It is concluded that in these cells, an altered architecture of the cortical network leads to a reduced overall stiffness of the cell, which is insufficient to support the force generation required for membrane deformation and pseudopod formation. Second, the role of cGMP in myosin II dynamics was investigated. Cyclic GMP is known to regulate the association of myosin II with the cytoskeleton. In Dictyostelium, intracellular cGMP levels increase when cells are exposed to chemoattractants, but also in response to osmotic stress. To study the influence of cyclic GMP on actin and myosin II dynamics, I used the laser-induced photoactivation of a DMACM-caged-Br-cGMP to locally release cGMP inside the cell. My results show that cGMP directly activates the myosin II machinery, but is also able to induce an actin response independently of cAMP receptor activation and signaling. The actin response was observed in both vegetative and developed cells. Possible explanations include cGMP-induced actin polymerization through VASP (vasodilator-stimulated phosphoprotein) or through binding of cGMP to cyclic nucleotide-dependent kinases. Finally, I investigated the role of phosphoinositide signaling using the Polyphosphoinositide-Binding Peptide (PBP10) that binds preferentially to PIP2. Phosphoinositides can recruit actin-binding proteins to defined subcellular sites and alter their activity. Neutrophils, as well as developed Dictyostelium cells produce PIP3 in the plasma membrane at their leading edge in response to an external chemotactic gradient. Although not essential for chemotaxis, phosphoinositides are proposed to act as an internal compass in the cell. When treated with the peptide PBP10, cells became round, with fewer or no pseudopods. PH-CRAC translocation to the membrane still occurs, even at low cAMP stimuli, but cell motility (random and directional) was reduced. My data revealed that the decrease in the pool of available PIP2 in the cell is sufficient to impair cell motility, but enough PIP2 remains so that PIP3 is formed in response to chemoattractant stimuli. My data thus highlights how sensitive cell motility and morphology are to changes in the phosphoinositide signaling. In summary, I have analyzed representative regulatory mechanisms that govern key parts of the motile machinery and characterized their impact on cellular properties including mechanical stability, adhesion and chemotaxis. N2 - Das Ziel der Arbeit war es, die regulatorischen Mechanismen der Zellmotilität zu untersuchen. Dazu habe ich für jedes Stadium dieses Prozesses einen repräsentativen regulatorischen Schritt ausgewählt und genauer untersucht: Die Regelung des Arp2/3 Komplexes durch WASP (Aktinsystem), die Rolle von cGMP in der Myosin II-Regulation (Myosinsystem) und der Einfluss von Phosphoinositiden im intrazellulären Signalprozess (Rezeptor-Signalweg). Die soziale Amöbe Dictyostelium discoideum wurde als Modellorganismus für diese Arbeiten gewählt. Gründe für diese Wahl waren die bereits vorliegenden detaillierten Kenntnisse über das Zytoskelett dieser Zellen, ihre einfache Handhabbarkeit im Labor, und die hohe Motilität der Zellen im vegetativen und entwickelten Zustand. Als Erstes analysierte ich die Dynamik des Aktin-Zytoskeletts durch Modulation der Aktivität des Arp2/3-Komplexes. Dafür benutzte ich das Carbazol-Derivat Wiskostatin, ein Inhibitor des Arp2/3-Aktivators WASP. Zellen, die mit Wiskostatin behandelt wurden, zeigten eine runde Form mit wenigen oder keinen Pseudopodien. Mit Hilfe des mikrofluidischen cell squeezer device konnte ich zeigen, dass Wiskostatin-behandelte Zellen eine geringere mechanische Stabilität aufweisen, vergleichbar mit Zellen unter dem Einfluss des Aktin-depolymerisierenden Wirkstoffes Latrunculin A. Darüber hinaus zeigen Wiskostatin behandelten Zellen eine erhöhte Substratadhäsion und eine verringerte Motilität und chemotaktische Effizienz. Der F-Aktingehalt der Zelle insgesamt blieb jedoch unverändert. Konfokal- und TIRF-mikroskopische Aufnahmen zeigten, dass die Zellen einen intakten Aktinkortex aufweisen. Es konnten lokalisierte dynamische Regionen erhöhter Aktinpolymerisation beobachtet werden, die jedoch nicht zur Ausbildung von Membrandeformationen führten. Daraus kann man rückschließen, dass die Mechanismen der Krafterzeugung im Aktin-Zytoskelett in WASP-inhibierten Zellen beeinträchtigt sind. Vermutlich liegt in diesen Zellen eine veränderte Mikroarchitektur des kortikalen Netzwerks vor, die zu einer verminderten Steifigkeit der Zelle führt, so dass die zur Bildung von Pseudopodien erforderlichen Kräfte nicht entfaltet werden können. Als Zweites wurde die Rolle von cGMP in der Myosin II-Dynamik untersucht. Es ist bekannt, dass cGMP die Assoziation von Myosin II mit dem Zytoskelett reguliert. In Dictyostelium steigt die intrazelluläre Konzentration von cGMP in Gegenwart von chemoattraktiven Lockstoffen sowie in Antwort auf osmotischen Stress. Um den Einfluss von cGMP auf die Aktin und Myosin II -Dynamik zu untersuchen, benutzte ich laserinduzierte Photoaktivierung von DMACM-caged-Br-cGMP, um cGMP lokal innerhalb der Zelle freizusetzen. Meine Ergebnisse zeigten, dass intrazelluläres cGMP direkt zur Aktivierung von Myosin II führt, jedoch auch Aktinantworten unabhängig vom cAMP-Rezeptorsignalweg induzieren kann. Die Aktinreaktion wurde sowohl in vegetativen als auch in entwickelten Zellen beobachtet. Eine mögliche Erklärung könnte die cGMP-induzierte Aktinpolymerisation über VASP (vasodilator-stimulated phosphoprotein) sein oder über die Bindung von cGMP an Nukleotid-abhängige Proteinkinasen. Als dritten Punkt meiner Arbeit untersuchte ich die Rolle der Phosphoinositide mit Hilfe des Phosphoinositide-bindenden Proteins PBP10, das bevorzugt an PIP2 bindet. Phosphoinositiden können Aktin-bindende Proteine zu definierten subzellulären Orten rekrutieren und ihre Aktivität verändern. Sowohl Neutrophile als auch entwickelte Dictyostelium Zellen produzieren PIP3 in der Plasmamembran an ihrer leading edge in Antwort auf externe Gradienten chemischer Lockstoffe. Obwohl Zellen auch ohne PIP3 chemotaktisches Verhalten zeigen, werden Phosphoinositide im Allgemeinen mit dem inneren chemotaktischen Kompass der Zelle in Verbindung gebracht. Mit dem Peptid PBP10 behandelte Zellen nahmen eine runde Form an, mit wenigen oder keinen Pseudopodien. PH-CRAC -Translokation zur Membran konnte in PBP10-behandelten Zellen selbst bei geringen cAMP-Stimuli weiterhin beobachtet werden. Ungerichtete wie auch gerichtete Zellmotiliät waren jedoch beeinträchtigt. Meine Daten zeigen, dass die Abnahme des PIP2-Pools in der Zelle durch PBP10 ausreicht, um die Zellmotilität zu beeinträchtigen, dass jedoch genug PIP2 erhalten bleibt um in Folge einer Rezeptorstimulation PIP3 zu produzieren. Die Ergebnisse demonstrieren daher, wie empfindlich Zellmotilität und -morphologie gegenüber Modifikationen im Phosphoinositid-Signalweg sind. Zusammenfassend habe ich mehrere repräsentative Beispiele für regulatorische Mechanismen der Zellmotilität untersucht und deren Auswirkung auf Eigenschaften der Zelle wie mechanische Stabilität, Zelladhäsion und Chemotaxis charakterisiert. KW - Dictyostelium KW - Aktomyosin KW - Wiskostatin KW - cGMP KW - PBP10 KW - Dictyostelium KW - actomyosin KW - Wiskostatin KW - cGMP KW - PBP10 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-57812 ER - TY - JOUR A1 - Batsios, Petros A1 - Gräf, Ralph A1 - Koonce, Michael P. A1 - Larochelle, Denis A. A1 - Meyer, Irene T1 - Nuclear envelope organization in Dictyostelium discoideum JF - The international journal of developmental biology N2 - The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export.The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun 1, as well as with the LEM/HeH-family protein Src1. Sun 1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun 1 usually forms a so-called LINC complex.Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in perm eabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics. KW - nuclear envelop KW - Dictyostelium KW - lamin KW - NET KW - centrosome KW - centromere Y1 - 2019 U6 - https://doi.org/10.1387/ijdb.190184rg SN - 0214-6282 SN - 1696-3547 VL - 63 IS - 8-10 SP - 509 EP - 519 PB - UBC Pr CY - Bilbao ER - TY - GEN A1 - Batsios, Petros A1 - Ren, Xiang A1 - Baumann, Otto A1 - Larochelle, Denis A. A1 - Gräf, Ralph T1 - Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81 N2 - The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 263 KW - Dictyostelium KW - HeH-protein KW - LEM-domain protein KW - lamin KW - nuclear lamina KW - nucleolus KW - nucleus Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-97033 ER - TY - JOUR A1 - Batsios, Petros A1 - Ren, Xiang A1 - Baumann, Otto A1 - Larochelle, Denis A. A1 - Gräf, Ralph T1 - Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81 JF - Cells N2 - The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture. KW - Dictyostelium KW - lamin KW - nuclear lamina KW - nucleus KW - nucleolus KW - HeH-protein KW - LEM-domain protein Y1 - 2016 U6 - https://doi.org/10.3390/cells5010013 SN - 2073-4409 VL - 5 IS - 1 PB - MDPI CY - Basel ER - TY - JOUR A1 - Gerhardt, Matthias A1 - Walz, Michael A1 - Beta, Carsten T1 - Signaling in chemotactic amoebae remains spatially confined to stimulated membrane regions JF - Journal of cell science N2 - Recent work has demonstrated that the receptor-mediated signaling system in chemotactic amoeboid cells shows typical properties of an excitable system. Here, we delivered spatially confined stimuli of the chemoattractant cAMP to the membrane of differentiated Dictyostelium discoideum cells to investigate whether localized receptor stimuli can induce the spreading of excitable waves in the G-protein-dependent signal transduction system. By imaging the spatiotemporal dynamics of fluorescent markers for phosphatidylinositol (3,4,5)-trisphosphate (PIP3), PTEN and filamentous actin, we observed that the activity of the signaling pathway remained spatially confined to the stimulated membrane region. Neighboring parts of the membrane were not excited and no receptor-initiated spatial spreading of excitation waves was observed. To generate localized cAMP stimuli, either particles that carried covalently bound cAMP molecules on their surface were brought into contact with the cell or a patch of the cell membrane was aspirated into a glass micropipette to shield this patch against freely diffusing cAMP molecules in the surrounding medium. Additionally, the binding site of the cAMP receptor was probed with different surface-immobilized cAMP molecules, confirming results from earlier ligand-binding studies. KW - Signal transduction KW - Excitable dynamics KW - Dictyostelium KW - cAMP KW - PIP3 KW - PIP2 KW - PI3K KW - PTEN KW - Micropipette aspiration KW - cAMP receptor KW - Patch clamp Y1 - 2014 U6 - https://doi.org/10.1242/jcs.161133 SN - 0021-9533 SN - 1477-9137 VL - 127 IS - 23 SP - 5115 EP - 5125 PB - Company of Biologists Limited CY - Cambridge ER - TY - GEN A1 - Grafe, Marianne A1 - Batsios, Petros A1 - Meyer, Irene A1 - Lisin, Daria A1 - Baumann, Otto A1 - Goldberg, Martin W. A1 - Gräf, Ralph T1 - Supramolecular Structures of the Dictyostelium Lamin NE81 T2 - Potsprint der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 682 KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina KW - expansion microscopy Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-425976 SN - 1866-8372 IS - 682 ER - TY - JOUR A1 - Grafe, Marianne A1 - Batsios, Petros A1 - Meyer, Irene A1 - Lisin, Daria A1 - Baumann, Otto A1 - Goldberg, Martin W. A1 - Gräf, Ralph T1 - Supramolecular Structures of the Dictyostelium Lamin NE81 JF - Cells N2 - Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina KW - expansion microscopy Y1 - 2019 U6 - https://doi.org/10.3390/cells8020162 SN - 2073-4409 VL - 8 IS - 2 PB - Molecular Diversity Preservation International 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 - Grafe, Marianne A1 - Hofmann, Phillip A1 - Batsios, Petros A1 - Meyer, Irene A1 - Gräf, Ralph T1 - In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH JF - Cells : open access journal N2 - We expressedDictyosteliumlamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-Delta NLS Delta CLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of theDictyosteliumlamin, they are likely relevant also for wild-type lamin. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina Y1 - 2020 U6 - https://doi.org/10.3390/cells9081834 SN - 2073-4409 VL - 9 IS - 8 PB - MDPI CY - Basel ER -