Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. 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 analysed 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 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. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.     

Nucleoplasmic beta-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations

Beta-actin, once thought to be an exclusively cytoplasmic protein, is now known to have important functions within the nucleus. Nuclear beta-actin associates with and functions in chromatin remodeling complexes, ribonucleic acid polymerase complexes, and at least some ribonucleoproteins. Proteins involved in regulating actin polymerization are also found in the interphase nucleus. We define the dynamic properties of nuclear actin molecules using fluorescence recovery after photobleaching. Our results indicate that actin and actin-containing complexes are reduced in their mobility through the nucleoplasm diffusing at approximately 0.5 microm2 s(-1). We also observed that approximately 20% of the total nuclear actin pool has properties of polymeric actin that turns over rapidly. This pool could be detected in endogenous nuclear actin by using fluorescent polymeric actin binding proteins and was sensitive to drugs that alter actin polymerization. Our results validate previous reports of polymeric forms of nuclear actin observed in fixed specimens and reveal that these polymeric forms are very dynamic.     

Sequence and comparative genomic analysis of actin-related proteins

Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4.     

Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling

Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome‐wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label‐free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co‐IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co‐migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co‐elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC–MS analysis for system‐wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.     

Nuclear spectrin-like proteins are structural actin-binding proteins in plants

Background information. Although actin is a relevant component of the plant nucleus, only three nuclear ABPs (actin‐binding proteins) have been identified in plants to date: cofilin, profilin and nuclear myosin I. Although plants lack orthologues of the main structural nuclear ABPs in animals, such as lamins, lamin‐associated proteins and nesprins, their genome does contain sequences with spectrin repeats and N‐terminal calponin homology domains for actin binding that might be distant relatives of spectrin. We investigated here whether spectrin‐like proteins could act as structural nuclear ABPs in plants. Results. We have investigated the presence of spectrins in Allium cepa meristematic nuclei by Western blotting, confocal and electron microscopy, using antibodies against α‐ and β‐spectrin chains that cross‐react in plant nuclei. Their role as nuclear ABPs was analysed by co‐immunoprecipitation and IF (immunofluorescence) co‐localization and their association with the nuclear matrix was investigated by sequential extraction of nuclei with non‐ionic detergent, and in low‐ and high‐salt buffers after nuclease digestion. Our results demonstrate the existence of several spectrin‐like proteins in the nucleus of onion cells that have different intranuclear distributions in asynchronous meristematic populations and associate with the nuclear matrix. These nuclear proteins co‐immunoprecipitate and co‐localize with actin. Conclusions. These results reveal that the plant nucleus contains spectrin‐like proteins that are structural nuclear components and function as ABPs. Their intranuclear distribution suggests that plant nuclear spectrin‐like proteins could be involved in multiple nuclear functions.     

WAVE forms hetero- and homo-oligomeric complexes at integrin junctions in Drosophila visualized by bimolecular fluorescence complementation

Dynamic actin polymerization drives a variety of morphogenetic events during metazoan development. Members of the WASP/WAVE protein family are central nucleation-promoting factors. They are embedded within regulatory networks of macromolecular complexes controlling Arp2/3-mediated actin nucleation in time and space. WAVE (Wiskott-Aldrich syndrome protein family verprolin-homologous protein) proteins are found in a conserved pentameric heterocomplex that contains Abi, Kette/Nap1, Sra-1/CYFIP, and HSPC300. Formation of the WAVE complex contributes to the localization, activity, and stability of the various WAVE proteins. Here, we established the Bimolecular Fluorescence Complementation (BiFC) technique in Drosophila to determine the subcellular localization of the WAVE complex in living flies. Using different split-YFP combinations, we are able to visualize the formation of the WAVE-Abi complex in vivo. We found that WAVE also forms dimers that are capable of forming higher order clusters with endogenous WAVE complex components. The N-terminal WAVE homology domain (WHD) of the WAVE protein mediates both WAVE-Abi and WAVE-WAVE interactions. Detailed localization analyses show that formation of WAVE complexes specifically takes place at basal cell compartments promoting actin polymerization. In the wing epithelium, hetero- and homooligomeric WAVE complexes co-localize with Integrin and Talin suggesting a role in integrin-mediated cell adhesion. RNAi mediated suppression of single components of the WAVE and the Arp2/3 complex in the wing further suggests that WAVE-dependent Arp2/3-mediated actin nucleation is important for the maintenance of stable integrin junctions.