58340
2020
2020
eng
6
3
3
article
MDPI
Basel
1
2020-07-01
2020-07-01
--
Dictyostelium cell fixation
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.
Methods and protocols
two simple tricks
10.3390/mps3030047
32630359
2409-9279
outputup:dataSource:PubMed:2020
47
WOS:000698975500002
Koonce, M (corresponding author), NYS Dept Hlth, Wadsworth Ctr, Div Translat Med, Albany, NY 12237 USA., michael.koonce@health.ny.gov; irina.tikhonenko@health.ny.gov; <br /> rgraef@uni-potsdam.de
National Science FoundationNational Science Foundation (NSF); [MCB-1510511]
Koonce, Michael
2023-03-14T09:30:59+00:00
sword
importub
filename=package.tar
129b1c84eaf48753af5e02ef46e48203
2934611-3
false
true
CC-BY - Namensnennung 4.0 International
Michael Koonce
Irina Tikhonenko
Ralph Gräf
eng
uncontrolled
Dictyostelium
eng
uncontrolled
cell fixation
eng
uncontrolled
microscopy
eng
uncontrolled
microtubule
eng
uncontrolled
cytoskeleton
Biowissenschaften; Biologie
Medizin und Gesundheit
Institut für Biochemie und Biologie
Referiert
Import
Gold Open-Access
DOAJ gelistet
57392
2020
2020
eng
14
8
9
article
MDPI
Basel
1
2020-08-04
2020-08-04
--
In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH
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.
Cells : open access journal
10.3390/cells9081834
32759812
2073-4409
outputup:dataSource:MDPI:2020
1834
WOS:000564724200001
Graf, R (corresponding author), Univ Potsdam, Dept Cell Biol, Karl Liebknecht Str 24-25, D-14476 Golm, Germany., mgrafe@uni-potsdam.de; phillip.hofmann@outlook.com; <br /> batsios@uni-potsdam.de; irene.meyer@uni-potsdam.de; <br /> rgraef@uni-potsdam.de
Deutsche Forschungsgemeinschaft (DFG) German Research Foundation (DFG); [GR1642/4-3]
Gräf, Ralph
2023-01-09T11:15:31+00:00
sword
importub
filename=package.tar
7b0ae2863e9d32088d36d078ac22b2b1
Gräf, Ralph
CC-BY - Namensnennung 4.0 International
Marianne Grafe
Phillip Hofmann
Petros Batsios
Irene Meyer
Ralph Gräf
eng
uncontrolled
lamin
eng
uncontrolled
NE81
eng
uncontrolled
Dictyostelium
eng
uncontrolled
nuclear envelope
eng
uncontrolled
nuclear lamina
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Import
Gold Open-Access
DOAJ gelistet
56748
2021
2021
eng
26
10
10
article
MDPI
Basel
1
2021-10-05
2021-10-05
--
The dictyostelium centrosome
The centrosome of Dictyostelium amoebae contains no centrioles and consists of a cylindrical layered core structure surrounded by a corona harboring microtubule-nucleating gamma-tubulin complexes. It is the major centrosomal model beyond animals and yeasts. Proteomics, protein interaction studies by BioID and superresolution microscopy methods led to considerable progress in our understanding of the composition, structure and function of this centrosome type. We discuss all currently known components of the Dictyostelium centrosome in comparison to other centrosomes of animals and yeasts.
Cells : open access journal
10.3390/cells10102657
34685637
2073-4409
outputup:dataSource:PubMed:2021
2657
WOS:000712350600001
Graef, R (corresponding author), Univ Potsdam, Dept Cell Biol, Karl Liebknecht Str 24-25, D-14476 Potsdam, Germany., rgraef@uni-potsdam.de; mgrafe@uni-potsdam.de; <br /> irene.meyer@uni-potsdam.de; mitic@uni-potsdam.de; <br /> valentin.pitzen@uni-potsdam.de
Deutsche Forschungsgemeinschaft (DFG)German Research Foundation (DFG) [GR1642/9-1, GR1642/11-1, ME3690/2-1]
Gräf, Ralph
2022-11-21T06:16:50+00:00
sword
importub
filename=package.tar
5af7e28ba2818b95ace8f03b63f96f6c
2661518-6
false
true
CC-BY - Namensnennung 4.0 International
Ralph Gräf
Marianne Grafe
Irene Meyer
Kristina Mitic
Valentin Pitzen
eng
uncontrolled
microtubule-organizing center
eng
uncontrolled
microtubule-organization
eng
uncontrolled
centrosome
eng
uncontrolled
Dictyostelium
eng
uncontrolled
mitosis
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Import
Gold Open-Access
DOAJ gelistet
56288
2021
eng
19
9
10
article
MDPI
Basel
1
--
2021-09-10
--
Cep192, a novel missing link between the centrosomal core and corona in Dictyostelium amoebae
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.
Cells : open access journal
10.3390/cells10092384
34572033
2073-4409
outputup:dataSource:PubMed:2021
2384
WOS:000699256700001
Meyer, I (corresponding author), Univ Potsdam, Dept Cell Biol, Karl Liebknecht Str 24-25, D-14476 Potsdam, Germany., valentin.pitzen@uni-potsdam.de; s.sander88@gmail.com; <br /> obaumann@uni-potsdam.de; rgraef@uni-potsdam.de; <br /> irene.meyer@uni-potsdam.de
Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [ME3690/2-1]; Deutsche Forschungsgemeinschaft (DFG)German Research Foundation (DFG) [INST 336/114-1 FUGG]
Meyer, Irene
2022-10-13T11:57:12+00:00
sword
importub
filename=package.tar
93bc8c5a48b63cf4a46a4ba5562fc002
2661518-6
CC-BY - Namensnennung 4.0 International
Valentin Pitzen
Sophia Sander
Otto Baumann
Ralph Gräf
Irene Meyer
eng
uncontrolled
Cep192
eng
uncontrolled
SPD-2
eng
uncontrolled
centrosome
eng
uncontrolled
Dictyostelium
eng
uncontrolled
microtubule-organization
eng
uncontrolled
MTOC
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Import
Gold Open-Access
DOAJ gelistet
55214
2017
2017
eng
119
130
12
96
article
Elsevier
Jena
1
2017-01-12
2017-01-12
--
CP39, CP75 and CP91 are major structural components of the Dictyostelium
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.
European journal of cell biology
10.1016/j.eicb.2017.01.004
28104305
0171-9335
1618-1298
wos:2017
WOS:000412150200004
Meyer, I; Graf, R (reprint author), Univ Potsdam, Inst Biochem & Biol, Dept Cell Biol, Karl Liebknecht Str 24-25,Haus 26, D-14476 Potsdam, Germany., irene.meyer@uni-potsdam.de; rgraef@uni-porsdam.de
2022-06-17T08:19:48+00:00
sword
importub
filename=package.tar
34ca817be97cbd9caebb62bd54a64cd3
false
true
Irene Meyer
Tatjana Peter
Petros Batsios
Oliver Kuhnert
Anne Krueger-Genge
Carl Camurca
Ralph Gräf
eng
uncontrolled
Dictyostelium
eng
uncontrolled
Mitosis
eng
uncontrolled
Microtubules
eng
uncontrolled
Centrosome
eng
uncontrolled
Nucleus
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Import
54533
2022
2021
eng
14
3
11
article
MDPI
Basel
1
2022-01-25
2021-12-31
--
Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum
Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
Cells
10.3390/cells11030407
2073-4409
35159217
Universität Potsdam
PA 2022_019
2038.77
<a href="https://doi.org/10.25932/publishup-54534">Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 1233</a>
WOS:000754912100001
2661518-6
Meyer, Irene
CC-BY - Namensnennung 4.0 International
Kristina Mitic
Marianne Grafe
Petros Batsios
Irene Meyer
eng
uncontrolled
nuclear pore complex
eng
uncontrolled
nucleoporins
eng
uncontrolled
semi-closed mitosis
eng
uncontrolled
centrosome
eng
uncontrolled
Dictyostelium
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Publikationsfonds der Universität Potsdam
Gold Open-Access
54534
2022
2022
eng
16
3
postprint
1
2022-03-29
2022-03-29
--
Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum
Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
10.25932/publishup-54534
urn:nbn:de:kobv:517-opus4-545341
1866-8372
Cells 11 (2022) 3, 407 DOI: 10.3390/cells11030407
<a href="http://publishup.uni-potsdam.de/54533">Bibliographieeintrag der Originalveröffentlichung/Quelle</a>
true
true
CC-BY - Namensnennung 4.0 International
Kristina Mitic
Marianne Grafe
Petros Batsios
Irene Meyer
Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
1233
eng
uncontrolled
nuclear pore complex
eng
uncontrolled
nucleoporins
eng
uncontrolled
semi-closed mitosis
eng
uncontrolled
centrosome
eng
uncontrolled
Dictyostelium
Biowissenschaften; Biologie
open_access
Institut für Biochemie und Biologie
Referiert
Green Open-Access
Universität Potsdam
https://publishup.uni-potsdam.de/files/54534/pmnr1233.pdf
53310
2018
2018
eng
17
4
7
article
MDPI
Basel
1
2018-04-23
2018-04-23
--
CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome
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.
Cells
10.3390/cells7040032
29690637
2073-4409
wos:2018
32
WOS:000435179500009
Meyer, I (reprint author), Univ Potsdam, Dept Cell Biol, Karl Liebknecht Str 24-25, D-14476 Potsdam, Germany., valentin.pitzen@uni-potsdam.de; askarzad@uni-potsdam.de; rgraef@uni-potsdam.de; irene.meyer@uni-potsdam.de
University of Potsdam
2022-01-06T09:32:16+00:00
sword
importub
filename=package.tar
232be3c088811ff155ba132c367759d1
Meyer, Irene
false
true
Valentin Pitzen
Sophie Askarzada
Ralph Gräf
Irene Meyer
eng
uncontrolled
centrosome
eng
uncontrolled
centriole
eng
uncontrolled
Dictyostelium
eng
uncontrolled
microtubules
eng
uncontrolled
mitosis
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Import
Gold Open-Access
DOAJ gelistet
52741
2020
2020
eng
14
8
9
article
MDPI
Basel
1
2020-08-04
2020-04-23
--
In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH
We expressed Dictyostelium lamin (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-∆NLS∆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 the Dictyostelium lamin, they are likely relevant also for wild-type lamin.
Cells
Universität Potsdam
<a href="https://doi.org/10.25932/publishup-52507">Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 1213</a>
Version of record
false
false
CC-BY - Namensnennung 4.0 International
Marianne Grafe
Phillip Hofmann
Petros Batsios
Irene Meyer
Ralph Gräf
eng
uncontrolled
lamin
eng
uncontrolled
NE81
eng
uncontrolled
Dictyostelium
eng
uncontrolled
nuclear envelope
eng
uncontrolled
nuclear lamina
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Gold Open-Access
52507
2020
2020
eng
16
8
postprint
1
2020-08-04
2020-04-23
--
In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH
We expressed Dictyostelium lamin (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-∆NLS∆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 the Dictyostelium lamin, they are likely relevant also for wild-type lamin.
Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
10.25932/publishup-52507
urn:nbn:de:kobv:517-opus4-525075
1866-8372
<a href="http://publishup.uni-potsdam.de/52741">Bibliographieeintrag der Originalveröffentlichung/Quelle</a>
1834
Version of record
Cells 2020, 9(8), 1834; https://doi.org/10.3390/cells9081834
true
true
CC-BY - Namensnennung 4.0 International
Marianne Grafe
Phillip Hofmann
Petros Batsios
Irene Meyer
Ralph Gräf
Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
1213
eng
uncontrolled
lamin
eng
uncontrolled
NE81
lat
uncontrolled
Dictyostelium
eng
uncontrolled
nuclear envelope
eng
uncontrolled
nuclear lamina
Biowissenschaften; Biologie
open_access
Institut für Biochemie und Biologie
Referiert
Green Open-Access
Universität Potsdam
https://publishup.uni-potsdam.de/files/52507/pmnr1213.pdf