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Analysis of supramolecular assemblies of NE81, the first lamin protein in a non-metazoan organism
(2019)
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.
Functional analyses of microtubule and centrosome-associated proteins in Dictyostelium discoideum
(2011)
Understanding the role of microtubule-associated proteins is the key to understand the complex mechanisms regulating microtubule dynamics. This study employs the model system Dictyostelium discoideum to elucidate the role of the microtubule-associated protein TACC (Transforming acidic coiled-coil) in promoting microtubule growth and stability. Dictyostelium TACC was localized at the centrosome throughout the entire cell cycle. The protein was also detected at microtubule plus ends, however, unexpectedly only during interphase but not during mitosis. The same cell cycle-dependent localization pattern was observed for CP224, the Dictyostelium XMAP215 homologue. These ubiquitous MAPs have been found to interact with TACC proteins directly and are known to act as microtubule polymerases and nucleators. This work shows for the first time in vivo that both a TACC and XMAP215 family protein can differentially localize to microtubule plus ends during interphase and mitosis. RNAi knockdown mutants revealed that TACC promotes microtubule growth during interphase and is essential for proper formation of astral microtubules in mitosis. In many organisms, impaired microtubule stability upon TACC depletion was explained by the failure to efficiently recruit the TACC-binding XMAP215 protein to centrosomes or spindle poles. By contrast, fluorescence recovery after photobleaching (FRAP) analyses conducted in this study demonstrate that in Dictyostelium recruitment of CP224 to centrosomes or spindle poles is not perturbed in the absence of TACC. Instead, CP224 could no longer be detected at the tips of microtubules in TACC mutant cells. This finding demonstrates for the first time in vivo that a TACC protein is essential for the association of an XMAP215 protein with microtubule plus ends. The GFP-TACC strains generated in this work also turned out to be a valuable tool to study the unusual microtubule dynamics in Dictyostelium. Here, microtubules exhibit a high degree of lateral bending movements but, in contrast most other organisms, they do not obviously undergo any growth or shrinkage events during interphase. Despite of that they are affected by microtubuledepolymerizing drugs such as thiabendazole or nocodazol which are thought to act solely on dynamic microtubules. Employing 5D-fluorescence live cell microscopy and FRAP analyses this study suggests Dictyostelium microtubules to be dynamic only in the periphery, while they are stable at the centrosome. In the recent years, the identification of yet unknown components of the Dictyostelium centrosome has made tremendous progress. A proteomic approach previously conducted by our group disclosed several uncharacterized candidate proteins, which remained to be verified as genuine centrosomal components. The second part of this study focuses on the investigation of three such candidate proteins, Cenp68, CP103 and the putative spindle assembly checkpoint protein Mad1. While a GFP-CP103 fusion protein could clearly be localized to isolated centrosomes that are free of microtubules, Cenp68 and Mad1 were found to associate with the centromeres and kinetochores, respectively. The investigation of Cenp68 included the generation of a polyclonal anti-Cenp68 antibody, the screening for interacting proteins and the generation of knockout mutants which, however, did not display any obvious phenotype. Yet, Cenp68 has turned out as a very useful marker to study centromere dynamics during the entire cell cycle. During mitosis, GFP-Mad1 localization strongly resembled the behavior of other Mad1 proteins, suggesting the existence of a yet uncharacterized spindle assembly checkpoint in Dictyostelium.
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.