Supramolecular Structures of the Dictyostelium Lamin NE81

Abstract

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.

Nuclear envelope organization in Dictyostelium discoideum

“…Both light and electron microscopy revealed reticular assemblies resembling those published for lamins in MEFs (Grafe et al, 2019;Shimi et al, 2015). Deconvolved ExM images revealed a close alignment of individual filaments in parallel arrangements of two to four filaments ( Fig.…”

“…As in metazoan lamins, an α-helical, central rod domain consisting of 370 amino acid residues is preceded by a head domain including Dictyostelium nuclear envelope 511 a CDK1 phosphorylation consensus sequence and followed by (in this order) a tail domain featuring a NLS, a conserved lamin tail domain, and a CaaX-box (= cysteine, two aliphatic aa and X = residue specifying the type of isoprene moiety) for prenylation at the C-terminal end (Krüger et al, 2012). Furthermore, microscopic and molecular studies confirmed that NE81 should be considered as a bona fide lamin due to its localization, functions, and protein interactions Batsios et al, 2016a;Batsios et al, 2016b;Grafe et al, 2019;Krüger et al, 2012). For example, immunogold-electron microscopy revealed an exclusive distribution of NE81 along the inner nuclear envelope.…”

“…Thus, the paradigm that intermediate filaments are part of a metazoan-specific protein toolkit was remained, until a lamin-like protein called NE81 was identified in Dictyostelium (Krüger et al, 2012). NE81 not only showed protein sequence similarity to metazoan lamins, it also behaved as a lamin from a functional point of view Grafe et al, 2019;Krüger et al, 2012). The identification of a lamin in one of the non-opisthokont branches of the eukaryotic tree of life and the improvement of bioinformatics tools facilitated identification of lamin-related protein sequences in organisms representing Archaeplastida, Excavata and SAR (Stramenopile, Alveolata, Rhizaria) (Kollmar, 2015;Preisner et al, 2018).…”

“…Recently, Grafe and co-workers have been successful in visualizing supramolecular NE81 filament assemblies by stimulated emission depletion microscopy (STED), Expansion microscopy (ExM) (Wassie et al, 2019) ( Fig. 2), transmission EM and feSEM (Grafe et al, 2019). They expressed Flag-tagged NE81 in Xenopus oocytes and showed by feSEM that NE81 assembles into filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2.…”

In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH

“…We were the first to discover a bona fide lamin in a non-metazoan organism [7]. In a series of experiments including mutational analyses and in vitro assembly studies, we could show that the Dictyostelium lamin NE81 is not only evolutionarily related to metazoan lamins and capable of forming filaments of approximately 10 nm in diameter, but also fulfills typical lamin functions [7][8][9][10]. Not surprisingly, NE81 behaved more as a B-type lamin [11].…”