New Insights into Cellular Functions of Nuclear Actin (original) (raw)
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Analysis of nuclear actin-interacting proteins and actin-regulated transcription factors
2019
Actin is best-known from its functions in the cytoplasm, where it is a key component of the cytoskeleton. Cytoskeleton is vital for cells as it enables cell movement and maintains cell shape. Nevertheless, functions of actin are not restricted to the cytoplasm, since actin is also present in the nucleus, where it has been linked to multiple functions from gene activation to chromatin remodeling. Live cell imaging with different nuclear actin probes have demonstrated the importance of actin dynamics inside the nucleus, but the molecular mechanisms by which actin operates in the nucleus are still poorly understood. This is mainly because of the lack of wellcharacterized binding partners for nuclear actin. Therefore, the aim of this thesis was to identify and characterize novel nuclear actin-binding partners and elucidate the molecular mechanisms behind actin regulated transcription factors. To identify nuclear actin-binding partners, we used two complementary mass spectrometry (MS) techniques, affinity purification combined with MS (AP-MS) and proximity dependent biotin identification with MS (BioID). AP-MS protocol was optimized to preserve complete nuclear complexes and BioID was geared towards identifying more transient interactions. We utilized different actin constructs to discriminate nuclear versus cytoplasmic interactions and to assess the requirement for actin polymerization for the putative nuclear interactions. Analysis of our interactome data revealed that actin can form stable complexes with proteins related to chromatin remodeling but seems to function in a dynamic fashion in other nuclear processes, such as transcription and DNA replication. In our experimental setup actin seemed to be monomeric when it associated with nuclear complexes. We also discovered a novel actin-containing complex, human Ada-Two-A-containing complex (hATAC). HATAC is a histone modifying complex and further studies showed that actin directly binds one of it subunits, lysine acetyltransferase 14 (KAT14). We showed that actin-binding modulates histone acetyl transferase (HAT) activity of KAT14 in vitro and in cells. We obtained numerous RNA splicing and mRNA processing factors with our BioID approach, which led us to investigate the role of actin in RNA splicing. Bimolecular fluorescent complementation (BiFC) assays demonstrated that actin associates with different splicing factors and we further showed, for the first time, that actin has a functional role in mRNA splicing, as alterations in nuclear actin levels disturbed survival motor neuron protein 2 (SMN2) alternative exon skipping. In addition, the nuclear actin interactome analysis provided new insights into nuclear processes already earlier linked to actin, such as chromatin remodeling, transcription and DNA replication, and hence this work provides a protein interaction platform for further mechanistic studies of nuclear actin-dependent functions. This thesis work has thereby broadened the knowledge of nuclear actin-binding partners as well as revealed novel regulatory properties of actin-regulated RPEL domain containing proteins.
Actin-related proteins in the nucleus: life beyond chromatin remodelers
Current Opinion in Cell Biology, 2010
Since their discovery in the mid-1990s, nuclear actin-related proteins (ARPs) have gained attention for their roles as structural components of ATP-dependent chromatinremodeling complexes. These remodelers can move nucleosomes along the DNA, evict them from chromatin, and exchange histone variants to alter chromatin states locally. Chromatin-remodeling facilitates DNA-templated processes such as transcription regulation, DNA replication, and repair. Consistent with a role for ARPs in shaping chromatin structure, recent genetic studies show that they affect developmental and cell-type specific transcriptional programming. Here, we focus on recent results that suggest a specific contribution of ARPs to long-range interactions in the nucleus, and review evidence indicating that some ARPs may act independently of chromatin-remodeling machines.
Nuclear actin levels as an important transcriptional switch
Transcription
Nuclear actin levels have recently been linked to different cellular fates, suggesting that actin could act as a switch between altered transcriptional states. Here we discuss our latest results on the mechanisms by which nuclear actin levels are regulated and their implications to the functional significance of nuclear actin.
Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro
Proceedings of the National Academy of Sciences, 2003
Structural protein 4.1, which has crucial interactions within the spectrin–actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher-resolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, a...
Nuclear Actin-Related Proteins as Epigenetic Regulators of Development
PLANT PHYSIOLOGY, 2005
Complex regulatory networks control cell fate and the development of organs and tissues in multicellular organisms. But what mechanisms initiate the necessary global changes in patterns of gene expression? What regulates the regulators of organismal development? The nuclear actin-related proteins (ARPs) par-
Nuclear Actin-Binding Proteins as Modulators of Gene Transcription
Traffic, 2005
Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.
Nuclear Actin and Actin-Binding Proteins in DNA Repair
Trends in Cell Biology, 2019
Nuclear actin has been implicated in a variety of DNA-related processes including chromatin remodeling, transcription, replication, and DNA repair. However, the mechanistic understanding of actin in these processes has been limited, largely due to a lack of research tools that address the roles of nuclear actin specifically, that is, distinct from its cytoplasmic functions. Recent findings support a model for homology-directed DNA double-strand break (DSB) repair in which a complex of ARP2 and ARP3 (actin-binding proteins 2 and 3) binds at the break and works with actin to promote DSB clustering and homology-directed repair. Further, it has been reported that relocalization of heterochromatic DSBs to the nuclear periphery in Drosophila is ARP2/3 dependent and actin-myosin driven. Here we provide an overview of the role of nuclear actin and actin-binding proteins in DNA repair, critically evaluating the experimental tools used and potential indirect effects. Nuclear Actin: Globular and Filamentous Forms Highlights Methods used to study nuclear actin have major caveats that can lead to biased data interpretation. Globular actin in nuclear remodeling complexes and histone acetyltransferases is often neglected due to a focus on filamentous nuclear actin. Recent evidence implicates nuclear actin and actin-binding proteins in resectiondependent DNA repair. The mechanism is unclear. The field needs further development of tools, the use of technical controls, and a systematic evaluation of alternative interpretations to reveal the function of nuclear actin.
Actin complexes in the cell nucleus: new stones in an old field
Histochemistry and Cell Biology, 2010
Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to structural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regulation of gene expression performed via concerting nuclear and cytoplasmic processes.
REVIEW Actin complexes in the cell nucleus: new stones in an old field
2010
Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to struc-tural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regu-lation of gene expression performed via concerting nuclear and cytoplasmic processes.
Embo Journal, 2011
Nuclear actin and actin related proteins (Arps) are integral components of various chromatin remodeling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeler. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analyzed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several intriguing differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments by complex formation with monomeric ADP-actin via the barbed end. Our data suggest a likely assembly between actin and Arp4, explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeler.