Actin and myosin are major components of the cell cytoskeleton, with structural and regulatory functions that affect many essential cellular processes. Although they were traditionally thought to ...function only in the cytoplasm, it is now well accepted that actin and multiple myosins are found in the nucleus. Increasing evidence on their functional roles has highlighted the importance of these proteins in the nuclear compartment.
One of the major cellular mechanisms to ensure cellular protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is triggered by accumulation of misfolded proteins in the ...ER lumen. The ER stress response is also activated in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we explore the mechanism of activation of the ER stress response in HGPS. We find that aggregation of the diseases-causing progerin protein at the nuclear envelope triggers ER stress. Induction of ER stress is dependent on the inner nuclear membrane protein SUN2 and its ability to cluster in the nuclear membrane. Our observations suggest that the presence of nucleoplasmic protein aggregates can be sensed, and signaled to the ER lumen, via clustering of SUN2. These results identify a mechanism of communication between the nucleus and the ER and provide insight into the molecular disease mechanisms of HGPS.
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•Aggregation of the progerin protein in the nucleus triggers ER stress•Induction of ER stress in progeria depends on the nuclear membrane protein SUN2•SUN2 forms progerin-induced protein clusters at the nuclear periphery•SUN2 clusters sequester ER chaperones and trigger an ER stress response
Vidak et al. find that aggregation of the disease-causing progerin protein at the nuclear periphery promotes sequestration of ER chaperones and induces ER stress. The inner nuclear membrane protein SUN2 senses progerin aggregation and transmits this information to the ER lumen, establishing an alternative mechanism to activate the ER stress response.
•A high-throughput imaging method for visualization of protein-protein interactions.•A panel of 60 lamin A/C interactors were identified.•Progerin alters interaction behavior of a subset of nuclear ...proteins.•Changes in protein interactions largely correlate with interactor expression.
Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the well-established antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize protein interactomes. Using HiPLA with a library of antibodies targeting nuclear proteins, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. Using HiPLA in combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions are accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.
Class I histone deacetylases (HDACs) are known to remove acetyl groups from histone tails. This liberates positive charges on the histone tail and allows for tighter winding of DNA, preventing ...transcription factor binding and gene activation. Although the functions of HDAC proteins are becoming apparent both biochemically and clinically, how this class of proteins is regulated remains poorly understood. We identified a novel interaction between nuclear actin and HDAC 1 and HDAC 2. Nuclear actin has been previously shown to interact with a growing list of nuclear proteins including chromatin remodeling complexes, transcription factors and RNA polymerases. We find that monomeric actin is able to bind the class I HDAC complex. Furthermore, increasing the concentration of actin in HeLa nuclear extracts was able to suppress overall HDAC function. Conversely, polymerizing nuclear actin increased HDAC activity and decreased histone acetylation. Moreover, the interaction between class I HDACs and nuclear actin was found to be activity dependent. Together, our data suggest nuclear actin is able to regulate HDAC 1 and 2 activity.
Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this ...question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.
α-Catenin is an F-actin-binding protein widely recognized for its role in cell-cell adhesion. However, a growing body of literature indicates that α-catenin is also a nuclear protein. In this study, ...we show that α-catenin is able to modulate the sensitivity of cells to DNA damage and toxicity. Furthermore, nuclear α-catenin is actively recruited to sites of DNA damage. This recruitment occurs in a β-catenin-dependent manner and requires nuclear actin polymerization. These findings provide mechanistic insight into the WNT-mediated regulation of the DNA damage response and suggest a novel role for the α-catenin-β-catenin complex in the nucleus.
Actin plays a crucial role in regulating multiple processes within the nucleus, including transcription and chromatin organization. However, the polymerization state of nuclear actin remains ...controversial, and there is no evidence for persistent actin filaments in a normal interphase nucleus. Further, several disease pathologies are characterized by polymerization of nuclear actin into stable filaments or rods. These include filaments that stain with phalloidin, resulting from point mutations in skeletal α-actin, detected in the human skeletal disease intranuclear rod myopathy, and cofilin/actin rods that form in response to cellular stressors like heatshock. To further elucidate the effects of these pathological actin structures, we examined the nucleus in both cell culture models as well as isolated human tissues. We find these actin structures alter the distribution of both RNA polymerase II and chromatin. Our data suggest that nuclear actin filaments result in disruption of nuclear organization, which may contribute to the disease pathology.
Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in ...the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus.
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•Adenovirus infection causes dramatic changes in the host's transcription machinery.•Nuclear actin is recruited to viral centers during the late phase of infection.•Nuclear myosin I is recruited with nuclear actin to viral replication centers.•Nuclear myosin V and VI are recruited to distinctive regions of viral centers.•Cytoplasmic and nuclear actin polymerization are necessary for Ad replication.
•Development of a single molecule tracking method for nuclear envelope proteins.•Measurement of single molecule dynamics of nuclear envelope proteins.•Pathological lamin A mutations differentially ...affect binding dynamics.•Lamin A mutants at the nuclear periphery and interior exhibit distinct dynamics.
The nuclear envelope (NE) is an essential cellular structure that contributes to nuclear stability, organization, and function. Mutations in NE-associated proteins result in a myriad of pathologies with widely diverse clinical manifestations, ages of onsets, and affected tissues. Notably, several hundred disease-causing mutations have been mapped to the LMNA gene, which encodes the intermediate filament proteins lamin A and C, two of the major architectural components of the nuclear envelope. However, how NE dysfunction leads to the highly variable pathologies observed in patient cells and tissues remains poorly understood. One model suggests alterations in the dynamic properties of the nuclear lamina and its associated proteins contribute to disease phenotype. Here, we describe the application of single molecule tracking (SMT) methodology to characterize the behavior of nuclear envelope transmembrane proteins and nuclear lamins in their native cellular environment at the single molecule level. As proof-of-concept, we demonstrate by SMT that Halo-tagged lamin B1, Samp1, lamin A, and lamin AΔ50 have distinct binding and kinetic properties, and we identify several disease-relevant mutants which exhibit altered binding dynamics. SMT is also able to separately probe the dynamics of the peripheral and the nucleoplasmic populations of lamin A mutants. We suggest that SMT is a robust and sensitive method to investigate the relationship between pathogenic mutations or cellular processes and protein dynamics at the NE.
The past decades have provided remarkable insights into how the eukaryotic cell nucleus and the genome within it are organized. The combined use of imaging, biochemistry and molecular biology ...approaches has revealed several basic principles of nuclear architecture and function, including the existence of chromatin domains of various sizes, the presence of a large number of non-membranous intranuclear bodies, non-random positioning of genes and chromosomes in 3D space, and a prominent role of the nuclear lamina in organizing genomes. Despite this tremendous progress in elucidating the biological properties of the cell nucleus, many questions remain. Here, we highlight some of the key open areas of investigation in the field of nuclear organization and genome architecture with a particular focus on the mechanisms and principles of higher-order genome organization, the emerging role of liquid phase separation in cellular organization, and the functional role of the nuclear lamina in physiological processes.
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