Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is crucial for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna-/-) MEFs, and all LaA constructs prevented increased nuclear envelope ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit emerin to the nuclear membrane in Lmna-/- MEFs. Our finding that tags impede some LaA functions but not others might explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.

Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.

Expansion microscopy (ExM) is a superresolution technique for fixed specimens that improves resolution of a given microscopy system approximately fourfold. The gain in resolution in ExM is not achieved by improvement of the resolution of the microscope itself but by isotropic expansion of the sample. To achieve this, the sample is cross-linked to an expandable gel matrix that swells approximately fourfold by incubation in water. We have applied the method to Dictyostelium amoebae and discuss the pros and cons of different labeling techniques in combination with pre- and post-expansion staining protocols.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.

Vascular endothelial cells line the inner surface of all blood vessels, where they are exposed to polarized mechanical forces throughout their lifespan. Both basal substrate interactions and apical blood flow-induced shear stress regulate blood vessel development, remodeling, and maintenance of vascular homeostasis. Disruption of these interactions leads to dysfunction and vascular pathologies, although how forces are sensed and integrated to affect endothelial cell behaviors is incompletely understood. Recently the endothelial cell nucleus has emerged as a prominent force-transducing organelle that participates in vascular mechanotransduction, via communication to and from cell-cell and cell-matrix junctions. The LINC complex, composed of SUN and nesprin proteins, spans the nuclear membranes and connects the nuclear lamina, the nuclear envelope, and the cytoskeleton. Here we review LINC complex involvement in endothelial cell mechanotransduction, describe unique and overlapping functions of each LINC complex component, and consider emerging evidence that two major SUN proteins, SUN1 and SUN2, orchestrate a complex interplay that extends outward to cell-cell and cell-matrix junctions and inward to interactions within the nucleus and chromatin. We discuss these findings in relation to vascular pathologies such as Hutchinson-Gilford progeria syndrome, a premature aging disorder with cardiovascular impairment. More knowledge of LINC complex regulation and function will help to understand how the nucleus participates in endothelial cell force sensing and how dysfunction leads to cardiovascular disease.

In eukaryotic cells, the nuclear envelope (NE) is a membrane partition between the nucleus and the cytoplasm to compartmentalize nuclear contents. It plays an important role in facilitating nuclear functions including transcription, DNA replication and repair. In mammalian cells, the NE breaks down and then reforms during cell division, and in interphase it is restored shortly after the NE rupture induced by mechanical force. In this way, the partitioning effect is regulated through dynamic processes throughout the cell cycle. A failure in rebuilding the NE structure triggers the mixing of nuclear and cytoplasmic contents, leading to catastrophic consequences for the nuclear functions. Whereas the precise details of molecular mechanisms for NE reformation during cell division and NE restoration in interphase are still being investigated, here, we mostly focus on mammalian cells to describe key aspects that have been identified and to discuss the crosstalk between them.

Lamin A/C (LMNA) is an important component of nuclear lamina. Mutations cause arrhythmia, heart failure, and sudden cardiac death. While LMNA-associated cardiomyopathy typically has an aggressive course that responds poorly to conventional heart failure therapies, there is variability in severity and age of penetrance between and even within specific mutations, which is poorly understood at the cellular level. Further, this heterogeneity has not previously been captured to mimic the heterozygous state, nor have the hundreds of clinical LMNA mutations been represented. Herein, we have overexpressed cardiopathic LMNA variants in HEK cells and utilized state-of-the-art quantitative proteomics to compare the global proteomic profiles of (1) aggregating Q353 K alone, (2) Q353 K coexpressed with WT, (3) aggregating N195 K coexpressed with WT, and (4) nonaggregating E317 K coexpressed with WT to help capture some of the heterogeneity between mutations. We analyzed each data set to obtain the differentially expressed proteins (DEPs) and applied gene ontology (GO) and KEGG pathway analyses. We found a range of 162 to 324 DEPs from over 6000 total protein IDs with differences in GO terms, KEGG pathways, and DEPs important in cardiac function, further highlighting the complexity of cardiac laminopathies. Pathways disrupted by LMNA mutations were validated with redox, autophagy, and apoptosis functional assays in both HEK 293 cells and in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) for LMNA N195 K. These proteomic profiles expand our repertoire for mutation-specific downstream cellular effects that may become useful as druggable targets for personalized medicine approach for cardiac laminopathies.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ-cell genes during differentiation and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we find that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ-cell genes into a silenced state and activating a group of oocyte genes and Nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, crosstalk between genome architecture and NPCs is essential for successful cell fate transitions.

The presence of farnesylated proteins at the inner nuclear membrane (INM), such as the Lamins or Kugelkern in Drosophila, leads to specific changes in the nuclear morphology and accelerated ageing on the organismal level reminiscent of the Hutchinson-Gilford progeria syndrome (HGPS). Farnesyl transferase inhibitors (FTIs) can suppress the phenotypes of the nuclear morphology in cultured fibroblasts from HGPS patients and cultured cells overexpressing farnesylated INM proteins. Similarly, FTIs have been reported to suppress the shortened lifespan in model organisms. Here, we report an experimental system combining cell culture and Drosophila flies for testing the activity of substances on the HGPS-like nuclear morphology and lifespan, with FTIs as an experimental example. Consistent with previous reports, we show that FTIs were able to ameliorate the nuclear phenotypes induced by the farnesylated nuclear proteins Progerin, Kugelkern, or truncated Lamin B in cultured cells. The subsequent validation in Drosophila lifespan assays demonstrated the applicability of the experimental system: treating adult Drosophila with the FTI ABT-100 reversed the nuclear phenotypes and extended the lifespan of experimentally induced short-lived flies. Since kugelkern-expressing flies have a significantly shorter average lifespan, half the time is needed for testing substances in the lifespan assay.