Role Of Actin Research Articles

Structural Polymorphism of F-Actin is Coupled with its Mechanical Properties

Actin plays a major role in many cellular processes including cell motility, cell division, endocytosis, and exocytosis. External and internal mechanical forces lead to cytoskeletal rearrangements in the cell. Recently, we proposed that actin filaments may be directly involved in mechanosensing. Single molecule experiments suggest that F-actin is stiff with respect to extension. We used cryo-electron microscopy and image analysis to examine variation in the axial rise (the distance between two adjacent actin protomers along the one-start left handed helix) within frozen-hydrated actin filaments. We show that F-actin can be found in both stretched and compressed structural states. The magnitude of such mechanical deformation is far beyond what has been suggested for F-actin, and the axial rise can vary from 25 to 30 Angstroms. We demonstrate that the structural state of the filament strongly correlates with the variability of the axial rise, and actin's stretching stiffness depends upon the structural state of the subdomain 2, just as we had previously shown for the bending stiffness. We show that actin binding proteins that cross-link adjacent actin protomers, such as cofilin and the actin binding domain 2 of fimbrin, dramatically reduce the variability of the axial rise within the decorated filaments. Our results demonstrate the coupling between the mechanical properties of F-actin and its structural state and provide a structural basis for actin's role in mechanosensing.

Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells

Keratin filaments impart resilience against mechanical extension of the cell. Despite the pathophysiological relevance of this function, very little is known about the mechanical properties of intermediate filaments in living cells and how these properties are modulated. We used keratin mutants that mimic or abrogate phosphorylation of keratin 8-serine(431) and keratin 18-serine(52) and investigated their effect on keratin tortuousness after cell stretch release in squamous cell carcinoma cells. Cells transfected with the wild-type keratins were used as controls. We can show that keratin dephosphorylation alters the stretch response of keratin in living cells since keratin tortuousness was abolished when phosphorylation of keratin18-serine(52) was abrogated. Additional experiments demonstrate that keratin tortuousness is not simply caused by a plastic overextension of keratin filaments because tortuousness is reversible and requires an intact actin-myosin system. The role of actin in this process remains unclear, but we suggest anchorage of keratin filaments to actin during stretch that leads to buckling on stretch release. Dephosphorylated keratin18-serine(52) might strengthen the recoil force of keratin filaments and hence explain the abolished buckling. The almost exclusive immunolabeling for phosphorylated keratin18-serine (52) in the cell periphery points at a particular role of the peripheral keratin network in this regard.

The NF-κB Signaling Protein Bcl10 Regulates Actin Dynamics by Controlling AP1 and OCRL-Bearing Vesicles

The protein Bcl10 contributes to adaptive and innate immunity through the assembly of a signaling complex that plays a key role in antigen receptor and FcR-induced NF-κB activation. Here we demonstrate that Bcl10 has an NF-κB-independent role in actin and membrane remodeling downstream of FcR in human macrophages. Depletion of Bcl10 impaired Rac1 and PI3K activation and led to an abortive phagocytic cup rich in PI(4,5)P(2), Cdc42, and F-actin, which could be rescued with low doses of F-actin depolymerizing drugs. Unexpectedly, we found Bcl10 in a complex with the clathrin adaptors AP1 and EpsinR. In particular, Bcl10 was required to locally deliver the vesicular OCRL phosphatase that regulates PI(4,5)P(2) and F-actin turnover, both crucial for the completion of phagosome closure. Thus, we identify Bcl10 as an early coordinator of NF-κB-mediated immune response with endosomal trafficking and signaling to F-actin remodeling.

Modeling Protein Structure & Function: Pencil Transferase

The interrelationship of structure and function is a key theme of biology. One important example of this relationship involves how the three-dimensional shapes of proteins are related to the roles they play at the cellular level. The treatment of protein structure in many introductory textbooks, however, is cursory and focuses on the relationships among primary, secondary, tertiary, and quaternary structure without a clear link to how these levels of organization contribute to functional aspects of the protein. This short and engaging hands-on activity helps reinforce the connection between the ultimate spatial conformation of a specific protein and its “job.” By exploring the structure and function of a hypothetical protein, students develop a conceptual understanding of how these important elements connect in real life (e.g., the roles of actin and myosin in muscle contraction, of proton pumps in cellular energy conversions, or of immunoglobulin proteins in helping an immune system fight off foreign invaders). Although this activity was designed for secondary-level students, it could also be modified for undergraduates. With the advent of free and readily accessible programs for viewing protein structures, as well as an increasing number of structure files and curated collections of proteins, biology instructors have at their disposal an array of examples of the striking beauty and diversity of proteins. A brief glossary and selected resources are listed at the end of this article. Pipe cleaners (a.k.a. chenille stems) are often used to …

Violaceols Function as Actin Inhibitors Inducing Cell Shape Elongation in Fibroblast Cells

Violaceol-I and -II were isolated from a fractionated library of marine-derived fungal metabolites. These compounds increased the calcium ion concentration inside the cell and caused F-actin aggregation in rat fibroblast 3Y1 cells within 3 h resulting in cell shape elongation. Calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited violaceol-I and -II induced F-actin aggregation in 3Y1 cells, and hence violaceol-I and -II act in a calcium dependent manner. Violaceol-I and -II inhibited G-actin polymerization in vitro in a dose-dependent manner and strongly associated with G-actin, at dissociation equilibrium constants of 1.44 × 10(-8) M and 2.52 × 10(-9) M respectively. Here we report the identification of a novel function of violaceol-I and -II as actin inhibitors. Violaceol-I and -II induced cell shape elongation through F-actin aggregation in 3Y1 fibroblasts. These compounds may give researchers new insights into the role of actin in tumorigenesis and lead to the development of additional anti-tumor drugs.

Fascin promotes filopodia formation independent of its role in actin bundling

Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is widely thought that this function involves fascin directly bundling actin filaments, which is controlled by an N-terminal regulatory serine residue. In this paper, by studying cellular processes in Drosophila melanogaster that require fascin activity, we identify a regulatory residue within the C-terminal region of the protein (S289). Unexpectedly, although mutation (S289A) of this residue disrupted the actin-bundling capacity of fascin, fascin S289A fully rescued filopodia formation in fascin mutant flies. Live imaging of migrating macrophages in vivo revealed that this mutation restricted the localization of fascin to the distal ends of filopodia. The corresponding mutation of human fascin (S274) similarly affected its interaction with actin and altered filopodia dynamics within carcinoma cells. These data reveal an evolutionarily conserved role for this regulatory region and unveil a function for fascin, uncoupled from actin bundling, at the distal end of filopodia.

Substrate-selective and Calcium-independent Activation of CaMKII by α-Actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

A pathogen’s journey in the host cell

Manipulation of the actin cytoskeleton is a commonly used process by which bacterial pathogens and viruses are able to neutralize host defense mechanisms and subvert them in order to replicate in a hostile environment. Diverse bacteria display a wide array of mechanisms of regulation of microfilaments to enter, move within or exit the host cell. A less studied subject is how pathogens may co-opt the actin cytoskeleton to disturb vesicle trafficking pathways, namely phagolysosomal fusion, and avoid degradation. In fact, although actin plays a role in endosomal trafficking and phagosome maturation, the knowledge on the exact mechanisms and additional players is still scarce. Recently, we found that the Legionella pneumophila virulence factor VipA is an actin nucleator, associates with actin filaments and early endosomes during infection, and interferes in yeast organelle trafficking pathways, suggesting it may be linking actin dynamics to endosome biogenesis. Further studies on this protein, together with work on other bacterial effectors, may help shed light in the role of actin in endosomal maturation.

Actin Cytoskeleton Rest Stops Regulate Anterograde Traffic of Connexin 43 Vesicles to the Plasma Membrane

The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown. We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking. High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 μm/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P<0.01) in de novo endogenous Cx43 delivery to cell-cell borders. In Langendorff-perfused mouse heart preparations, Cx43/β-actin complexing is disrupted during acute ischemia, and inhibition of actin polymerization is sufficient to reduce levels of Cx43 gap junctions at intercalated discs. Actin is a necessary component of the cytoskeleton-based forward trafficking apparatus for Cx43. In cardiomyocytes, Cx43 vesicles spend a majority of their time pausing at nonsarcomeric actin rest stops when not undergoing microtubule-based transport to the plasma membrane. Deleterious effects on this interaction between Cx43 and the actin cytoskeleton during acute ischemia contribute to losses in Cx43 localization at intercalated discs.

176 CYTOCHALASIN PREVENTS SPERM HEAD INCORPORATION INTO FERTILIZED PIG OOCYTES

At fertilization, an acrosome-reacted sperm fuses to the plasma membrane of the oocyte. It is then internalized into the ooplasm and its head forms the male pronucleus that moves toward the female pronucleus. Experiments using cytochalasin, an inhibitor of actin polymerization, suggest that filamentous actin is necessary for spermatozoa incorporation deep into the oocyte cytoplasm. However, the exact role of actin in the process is not entirely clear and the consequences of inhibiting actin polymerization on embryo development are not known. In the present study, we investigated the effect of cytochalasin B on fertilization and subsequent embryo development in the porcine model. In vitro-matured oocytes were rinsed in fertilization medium, a modified Tris-buffered medium supplemented with 0.1% BSA and 1 mM caffeine. Groups of 30 oocytes were placed into 50-μL droplets of the medium covered with mineral oil. Fresh semen was collected from both a wild-type boar and a transgenic boar heterozygous for the green fluorescent protein (GFP), washed 3 times in Dulbecco's PBS and added at a final concentration of 5 × 105 cells mL–1 to each droplet containing the oocytes. The gametes were co-incubated for 5 h at 39°C under 5% CO2 in air. In the treatment group, 10 μg mL–1 of cytochalasin B was supplemented into the fertilization droplets; control groups received an equivalent amount of dimethyl sulfoxide, the solvent used to dissolve the inhibitor. After gamete co-incubation, potential zygotes were transferred to PZM-3 medium for culture. In experiment 1, mature oocytes were fertilized with the wild-type boar sperm. Embryos at the 6- or 8-cell stage were then collected, the blastomeres were separated and DNA from each blastomere was isolated for PCR analysis to monitor the presence of the male-specific SRY gene. In experiment 2, the GFP transgenic sperm was used for fertilization. Fertilized oocytes were cultured for 7 days and the resultant embryos were examined for GFP expression using an epifluorescence microscope. The developmental stages of the embryos were also determined by staining their nuclei with Hoechst 33342. We found that after cytochalasin B treatment, only 19.1% (21/110) of the blastomeres were positive for the SRY gene, whereas in the control group, SRY was detected in 54.5% (61/112) of the cases. In addition, in the cytochalasin B-treated group, 1.9% (7/376) of the embryos expressed GFP, as indicated by green fluorescence; this percentage was 35.4% (146/412) in the control embryos. The frequency of 2-cell and blastocyst-stage embryos was similar between the control and cytochalasin B-treated groups (84.1 vs 81.0%; and 11.6 vs 12.5%, respectively). The results indicate that the presence of cytochalasin B during fertilization effectively blocks the incorporation of the male nucleus into the newly formed zygote without affecting the developmental potential of the pre-implantation embryo.

Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.

Dynamin A, Myosin IB and Abp1 Couple Phagosome Maturation to F‐Actin Binding

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.

The vital role of actin and the intermediate filaments in the maintenance of cellular architecture

The cytoskeleton is a system of structural protein primarily involved in the cellular architectural maintenance, intracellular transport, cell division and movement. The classical major components of the cytoskeleton are microtubules, intermediate filaments and microfilaments. Traditionally the microtubules are involved in intracellular transport and organelle structural support. Intermediate filaments provide a rigid structural support to the cell while the microfilaments maintain the cellular surface architecture and support movement and contraction. Emerging evidences however are challenging this classical view of cytoskeleton functions which calls for further investigation. The present study investigates the structural role of the three classical cytoskeletal elements in the maintenance of cell shape and architecture. The study exploits the high resolving power of the confocal microscopy in displaying subcellular components of intact cells, coupled with differential interference imaging. The use of monoclonal antibodies in immunocytochemical staining increases the detection efficiency of the structural details of the cytoskeleton. The present study has empirically demonstrated that actin filaments provide a crucial mechanical support and maintain its contents in space. Intermediate filaments, while they contribute to the cell architectural support they typically sustain the nucleus and associated structures in spatial proximity inside the cell. Microtubule filaments exhibit no apparent involvement in cellular architecture maintenance. This study has highlighted the specific structural functions of the classical cytoskeletal elements in which actin filaments play the most important role. These results are important in such areas as cancer research and in pathogen entry and internalization into the cell. Key words: actin, intermediate filaments, microtubules, cell architecture, confocal microscopy

Adjudin disrupts spermatogenesis via the action of some unlikely partners

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a potent analog of lonidamine [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] known to disrupt germ cell adhesion, most notably elongating and elongated spermatids, in the seminiferous epithelium of adult rat testes and thus, leads to infertility in rats. Since the population of spermatogonia and spermatogonial stem cells (SSCs) in the seminiferous tubules is not significantly reduced by the treatment of rats with adjudin, adjudin-induced infertility is highly reversible, which enables reinitiation of spermatogenesis and germ cell re-population of the voided seminiferous epithelium. Furthermore, adjudin appears to exert its effects at the testis-specific atypical adherens junction (AJ) type known as ectoplasmic specialization (ES), most notably the apical ES at the Sertoli cell-spermatid interface. Thus, the hypothalamic-pituitary-gonadal axis is not unaffected and systemic side-effects are minimal. This also makes adjudin a potential candidate for male contraceptive development. Herein, we critically evaluate recent findings in the field and provide an updated model regarding the mechanism underlying adjudin-induced apical ES disruption. In short, adjudin targets actin filament bundles at the apical ES, the hallmark ultrastructure of this testis-specific junction type not found in any other epithelia/endothelia in mammals, by suppressing the expression of Eps8 (epidermal growth factor receptor pathway substrate 8), an actin capping protein that also plays a role in actin bundling, so that actin filament bundles can no longer be maintained at the apical ES. This is concomitant with a mis-localization of Arp3 (actin-related protein 3, a component of the Arp2/3 complex that induces actin nucleation/branching) recruited by drebrin E, causing "unwanted" actin branching, further destabilizing actin filament bundles at the apical ES. Additionally, adjudin blocks the expression of PAR6 (partitioning defective protein 6) and 14-3-3 (also known as PAR5) considerably at the apical ES, disrupting the homeostasis of endocytic vesicle-mediated protein trafficking, which in turn leads to an increase in protein endocytosis. The net result of these changes destabilizes cell adhesion and induces degeneration of the apical ES, causing premature release of spermatids, mimicking spermiation.

Fibrillar actin in nuclear apparatus of ciliate Paramecium Caudatum

Ciliates are a good model for studying the role of actin in the nuclear spatial organization and transcription activity due to their nuclear dualism. Actin localization in the nuclear apparatus of the ciliate Paramecium caudatum was studied using fluorescently labeled phalloidin and indirect immunocytochemistry. Fibrillar actin was visualized in both nuclei. Actin was revealed in areas of the chromatin that are often associated with the periphery of the amplified nucleoli in the macronucleus. Actin distribution depended on the cell physiological state. The infection of the macronucleus with the intranuclear endobiont Holospora obtusa resulted in a loss of nuclear actin, nuclear fragility, and redistribution of the phosphorylated form of the actinbinding protein cofilin. A spherical body that resembles the karyosphere was found in the macronuclear anlagen.

Myosin 1 controls membrane shape by coupling F-Actin to membrane

Cellular functions are intimately associated with rapid changes in membrane shape. Different mechanisms interfering with the lipid bilayer, such as the insertion of proteins with amphipatic helices or the association of a protein scaffold, trigger membrane bending. By exerting force on membranes, molecular motors can also contribute to membrane remodeling. Previous studies have shown that actin and myosin 1 participate in the invagination of the plasma membrane during endocytosis while kinesins and dynein with microtubules provide the force to elongate membrane buds at recycling endosomes and at the trans-Golgi network (TGN). Using live cell imaging we have recently shown that a myosin 1 (myosin 1b) regulates the actin dependent post-Golgi traffic of cargo and generates force that controls the assembly of F-actin foci and promotes with the actin cytoskeleton the formation of tubules at the TGN. Our data provide evidence that actin and myosin 1 can regulate membrane remodeling of organelles as well as having an unexpected role in the spatial organization of the actin cytoskeleton. Here, we discuss our results together with the role of actin and other myosins that have been implicated in the traffic of cargo.

Cell Biology: Actin Keeps Endocytosis on a Short Leash

High-resolution structural analysis of branched actin networks at the sites of clathrin-mediated endocytosis sheds light on the role of actin in endocytosis and mechanisms controlling actin assembly.

The role of actin in capacitation-related signaling: an in silico and in vitro study

BackgroundThe signalling cascades involved in many biological processes require the coordination of different subcellular districts. It is the case of the pathways involved in spermatozoa acquisition of fertilizing ability (the so called "capacitation"). In the present work the coordination of subcellular signalling, during the boar sperm capacitation, was studied by a computational and experimental approach. As first the biological network representing all the molecular interactions involved in capacitation was build and analyzed, then, an experimental set up was carried out to confirm the computational model-based prediction.ResultsThe analysis of computational model pointed out that the "actin polymerization" node had some important and unique features:- it is one of the most connected nodes,- it links in a specific manner all the intracellular compartments,- its removal from the network did not affect the global network topology but caused the loss of five important nodes (and among them the "plasma membrane" and "outer acrosome membrane" fusion).Thus, it was suggested that actin polymerization could be involved in the signaling coordination of different subcellular districts, and that its functional ablation could compromise spermatozoa ability to complete the capacitation (while the main signaling pathway remained unaffected). The experiments, carried out inhibiting the actin polymerization in capacitating boar spermatozoa by the administration of cytocalasin D (CD), demonstrated that the CD treatment inhibited spermatozoa ability to reach the full fertilizing ability, while, the examined signaling pathways (membrane acquisition of chlortetracicline pattern C, protein tyrosine phosphorylation, phospholipase C-γ1 relocalization, intracellular calcium response to zonae pellucidae) remained effective, thus, confirming the model-based hypothesis.ConclusionsThe model based-hypothesis was confirmed by the reported data obtained with the in vitro experiments, which strengthen the idea that the actin cytoskeleton is not only a mechanical support for the cell, but that it exerts a key role in signaling during the sperm capacitation.

The cytoskeleton and nucleus: the role of actin as a modulator of neuronal gene expression

Abstract Actin, arranged for example in stress fibres, provides a fundamental cytoskeletal frame­work function to all cell types. Notably, there is now mounting evidence that, in addition to cytoplasmic cytoskeletal regulation, ac­tin treadmilling provides a signal modulat­ing nuclear gene expression. In altering gene regulation, cytoplasmic and most likely also a nucleus-resident actin provides an addition­al (gene) regulatory twist to cell motility. So far, the transcription factor serum response factor (SRF) alongside its myocardin-relat­ed transcription factor (MRTF) cofactors has emerged as the main target of actin dynam­ics. In this review, we discuss the impact of actin signalling on nuclear gene expression in the nervous system, where the actin-MRTF-SRF module contributes to various processes including neuronal motility.

New nuclear partners for nucleosome assembly protein 1: unexpected associations

Histone chaperones are important players in chromatin dynamics. They are instrumental in nucleosome assembly and disassembly and in histone variant exchange reactions that occur during DNA transactions. The molecular mechanisms of their action are not well understood and may involve interactions with various protein partners in the context of the nucleus. In an attempt to further elucidate nuclear roles of histone chaperones, we performed a proteomic search for nuclear partners of a particular histone chaperone, nucleosome assembly protein 1 (Nap1). Proteins recognized as Nap1 partners by immuno-affinity capture and Far Western blots were identified by mass spectrometry. The identified partners are known to participate in a number of nuclear processes, including DNA replication, recombination, and repair as well as RNA transcription and splicing. Finding nuclear actin among the Nap1 partners may be of particular significance, in view of actin's role in transcription, transcription regulation, and RNA splicing. We are proposing a model of how actin-Nap1 interaction may be involved in transcription elongation through chromatin. In addition, awareness of the interactions between Nap1 and Hsp70, another identified partner, may help to understand nucleosome dynamics around sites of single-strand DNA break repair. These studies represent a starting point for further investigation of Nap1 associations in human cells.