Changes In Nuclear Structure Research Articles

Emerging views of the nucleus as a cellular mechanosensor.

The ability of cells to respond to mechanical forces is critical for numerous biological processes. Emerging evidence indicates that external mechanical forces trigger changes in nuclear envelope structure and composition, chromatin organization and gene expression. However, it remains unclear if these processes originate in the nucleus or are downstream of cytoplasmic signals. Here we discuss recent findings that support a direct role of the nucleus in cellular mechanosensing and highlight novel tools to study nuclear mechanotransduction.

Nup153 Interacts with Sox2 to Enable Bimodal Gene Regulation and Maintenance of Neural Progenitor Cells

Neural progenitor cells (NeuPCs) possess a unique nuclear architecture that changes during differentiation. Nucleoporins are linked with cell-type-specific gene regulation, coupling physical changes in nuclear structure to transcriptional output; but, whether and how they coordinate with key fate-determining transcription factors is unclear. Here we show that thenucleoporin Nup153 interacts with Sox2 in adultNeuPCs, where it is indispensable for their maintenance and controls neuronal differentiation. Genome-wide analyses show that Nup153 and Sox2 bind and co-regulate hundreds of genes. Binding of Nup153 to gene promoters or transcriptional end sites correlates with increased or decreased gene expression, respectively, and inhibiting Nup153 expression alters open chromatin configurations at its target genes, disrupts genomic localization of Sox2, and promotes differentiation invitro and a gliogenic fate switch invivo. Together, these findings reveal that nuclear structural proteins may exert bimodal transcriptional effects to control cell fate.

Histological, Immunohistochemical, and Biochemical Study of Experimentally Induced Fatty Liver in Adult Male Albino Rat and the Possible Protective Role of Pomegranate.

Nonalcoholic fatty liver disease is a major health problem and is considered the most common worldwide liver disease. Pomegranate has many biological activities and could modify the risk of hypercholesterolemia. The objective of the current research was to study the histological changes of experimentally induced fatty liver and possible protection by pomegranate. For this purpose, 50 adult male albino rats were divided into four groups, control group, pomegranate treated group that were given pomegranate juice for six weeks, fatty liver induced group that were fed on high fat diet for six weeks and protective group that were fed on high fat diet and received pomegranate juice for six weeks. Histological changes were detected in the fatty liver induced group in the form of disturbed hepatic architecture, dilatation and congestion of central veins, blood sinusoids and portal veins. Most of hepatocytes showed variable degrees of cytoplasmic vacuolation, mitochondrial structural changes, dilatation of endoplasmic reticulum in addition to nuclear structural changes like condensed chromatin, irregular shrunken nuclei and vacuolated nuclei. All these changes were associated with inflammatory cellular infiltrations, deposition of collagen fibers around the central vein, blood sinusoids, portal areas and in between the hepatocytes in addition to significant increase in number of hepatic stellate cells that was proved by electron microscope and confirmed by immunohistochemical study. Moreover, these structural changes were much less pronounced in animals treated with pomegranate either with or before receiving high fat diet. These findings suggested that pomegranate has a protective effect against experimentally induced fatty liver.

Lifetime measurements in shape transition nucleus 188Pt

Nuclear level lifetimes of high spin states in yrast and non-yrast bands of 188Pt nucleus have been measured using recoil distance plunger setup present at IUAC, Delhi. In the experiment nuclear states of interest were populated via 174Yb(18O,4n)188Pt reaction at a beam energy of 79MeV provided by 15 UD Pelletron accelerator. The extracted $B(E2\downarrow)$ values show an initial rise up to $4^{+}$ state and then a nearly constant behavior with spin along yrast band, indicating change of nuclear structure in 188Pt at low spins. The good agreement between experimental and TPSM model $B(E2\downarrow)$ values up to $ 4^+$ state suggests an increase in axial deformation of the nucleus. The average absolute $\beta_{2} = 0.20 (3)$ obtained from measured $B(E2\downarrow)$ values matches well the values predicted by CHFB and IBM calculations for oblate ( $ \beta_{2} \sim -0.19$ ) and prolate ( $\beta_{2} \sim 0.22$ ) shapes. As the lifetime measurements do not yield the sign of $ \beta_{2}$ , no definite conclusion can be drawn on the prolate or oblate collectivity of 188Pt on the basis of present measurements.

Genomic instability during reprogramming by nuclear transfer is DNA replication dependent

Somatic cells can be reprogrammed to a pluripotent state by nuclear transfer into oocytes, yet developmental arrest often occurs. While incomplete transcriptional reprogramming is known to cause developmental failure, reprogramming also involves concurrent changes in cell cycle progression and nuclear structure. Here we study cellular reprogramming events in human and mouse nuclear transfer embryos prior to embryonic genome activation. We show that genetic instability marked by frequent chromosome segregation errors and DNA damage arise prior to, and independent of, transcriptional activity. These errors occur following transition through DNA replication and are repaired by BRCA1. In the absence of mitotic nuclear remodelling, DNA replication is delayed and errors are exacerbated in subsequent mitosis. These results demonstrate that independent of gene expression, cell-type-specific features of cell cycle progression constitute a barrier sufficient to prevent the transition from one cell type to another during reprogramming.

The study of structure in 224–234 thorium nuclei within the framework IBM

An investigation has been made of the behaviour of nuclear structure as a function of an increase in neutron number from 224 Th to 234 Th. Thorium of mass number 234 is a typical rotor nucleus that can be explained by the SU(3) limit of the interacting boson model(IBM) in the algebraic nuclear model. Furthermore, 224−232 Th lie on the path of the symmetry-breaking phase transition. Moreover, the nuclear structure of 224 Th can be explained using X(5) symmetry. However, as 226−230 Th nuclei are not fully symmetrical nuclei, they can be represented by adding a perturbed term to express symmetry breaking. Through the following three calculation steps, we identified the tendency of change in nuclear structure. Firstly, the structure of 232 Th is described using the matrix elements of the Hamiltonian and the electric quadrupole operator between basis states of the SU(3) limit in IBM. Secondly, the low-lying energy levels and E2 transition ratios corresponding to the observable physical values are calculated by adding a perturbed term with the first-order Casimir operator of the U(5) limit to the SU(3) Hamiltonian in IBM. We compared the results with experimental data of 224−234 Th. Lastly, the potential of the Bohr Hamiltonian is represented by a harmonic oscillator, as a result of which the structure of 224−234 Th could be expressed in closed form by an approximate separation of variables. The results of these theoretical predictions clarify nuclear structure changes in Thorium nuclei over mass numbers of practical significance.

Critical Roles of Α-Type Lamins in Neutrophil Differentiation Revealed By Analyses of Lmna Trancriptional Regulation and Myeloid Models with Aberrant Lamin Α Expression

Neutrophils differentiate from common myeloid progenitors in the bone marrow where they form uniquely lobulated nuclei while acquiring multiple functions critical to their innate immune responses. We are investigating the roles of nuclear envelope (NE)-associated proteins, including the lamin B receptor (Lbr) and the nuclear lamina proteins lamin A/C, in regulating these nuclear changes, along with determining if these proteins are important to neutrophil functions including chemotaxis, phagocytosis, or reactive oxygen species (ROS) production. We previously reported that Lbr expression increases during neutrophil differentiation while lamin A/C expression decreases. We also used immortalized mouse myeloid progenitors to show that either loss of Lbr or increased lamin A/C expression caused nuclear hypolobulation similar to Pelger-Huët anomaly in humans, plus aberrant chemotaxis. Interestingly, cells lacking Lbr produced less ROS, whereas those overexpressing the lamins exhibited deficient phagocytosis. These studies suggest that the functions of these nuclear proteins go beyond simply supporting nuclear structural changes. Since our previous studies relied on the use of immortalized cell lines, which may not exhibit the full spectrum of normal neutrophil functional responses, we have focused on using techniques to derive and characterize neutrophils with aberrant NE protein expression from ex vivo cultured bone marrow. We also examined whether loss of both A-type lamins affects myeloid progenitor growth and differentiation.Here we show that functionally active neutrophils can be generated by ex vivo culture of bone marrow progenitors from cryopreserved stocks. Imaging flow cytometry, which combines the spatial resolution of fluorescence microscopy with the high throughput format of flow cytometry, was used to demonstrate that the ex vivo cultures at progressive stages of neutrophil maturation exhibit characteristic changes to cell surface marker expression. Customized gating and masking strategies were applied using the image analysis software to quantitatively measure phagocytosis of Escherichia coli particles and release of neutrophil extracellular traps (NETs). We also identified a method to evaluate NET release as a function of cell activation during phagocytosis.We next transduced our ex vivo cultured progenitors with retroviral vectors providing ectopic lamin A expression, and found that derived neutrophils exhibited hypolobulated nuclei morphologically resembling our in vitro models. We also knocked out Lmna expression in the immortalized myeloid progenitors by CRISPR-Cas9 mediated gene disruption, and identified defective growth of the progenitors plus aberrant ROS production in derived neutrophils, similar to the phenotypes observed in cells lacking Lbr.The transcriptional control of the Lmna gene during neutrophil differentiation also was investigated. In contrast to our previous report showing that the ETS factor GABP cooperates with PU.1 or C/EBPε to regulate the Lbr promoter, none of these factors affected background levels of Lmna promoter activities in COS cell reporter assays despite identified ETS and C/EBP consensus binding sites. However, addition of C/EBPα expression caused a greater than 50% decrease in Lmna promoter activities. Furthermore, quantitative ChIP assays demonstrated that C/EBPα most efficiently bound to identified C/EBP binding sequences in the Lmna promoter, indicating that C/EBPα inhibits Lmna expression in differentiating neutrophils.Combined our results demonstrate that precisely regulated levels of NE-associated proteins are critical to neutrophil nuclear maturation and their acquisition of certain functional responses. Our continued studies will utilize imaging flow cytometry to characterize the functions of ex vivo culture-derived neutrophils with aberrant A-type lamin expression, as well as identify roles of additional NE-associated proteins during neutrophil maturation. We are also poised to further characterize the control of genes encoding such NE proteins by various combinations of ETS and C/EBP transcription factors known to regulate myelopoiesis. We predict these studies will provide important insight into the molecular mechanisms governing neutrophil nuclear changes, and why aberrant lobulation is commonly associated with certain hematopoietic disorders. DisclosuresNo relevant conflicts of interest to declare.

Changes in nuclear structure along the Mn isotopic chain studied via charge radii

The hyperfine spectra of $^{51,53-64}$Mn were measured in two experimental runs using collinear laser spectroscopy at ISOLDE, CERN. Laser spectroscopy was performed on the atomic $3d^5\ 4s^2\ ^{6}\text{S}_{5/2}\rightarrow 3d^5\ 4s4p\ ^{6}\text{P}_{3/2}$ and ionic $3d^5\ 4s\ ^{5}\text{S}_2 \rightarrow 3d^5\ 4p\ ^{5}\text{P}_3$ transitions, yielding two sets of isotope shifts. The mass and field shift factors for both transitions have been calculated in the multiconfiguration Dirac-Fock framework and were combined with a King plot analysis in order to obtain a consistent set of mean-square charge radii which, together with earlier work on neutron-deficient Mn, allow the study of nuclear structure changes from $N=25$ across $N=28$ up to $N=39$. A clear development of deformation is observed towards $N=40$, confirming the conclusions of the nuclear moments studies. From a Monte Carlo Shell Model study of the shape in the Mn isotopic chain, it is suggested that the observed development of deformation is not only due to an increase in static prolate deformation but also due to shape fluctuations and triaxiality. The changes in mean-square charge radii are well reproduced using the Duflo-Zuker formula except in the case of large deformation.

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription.

Effective Cellular Morphology Analysis for Differentiation Processes by a Fluorescent 1,3a,6a-Triazapentalene Derivative Probe in Live Cells.

Nuclear and cytoplasmic morphological changes provide important information about cell differentiation processes, cell functions, and signal responses. There is a strong desire to develop a rapid and simple method for visualizing cytoplasmic and nuclear morphology. Here, we developed a novel and rapid method for probing cellular morphological changes of live cell differentiation process by a fluorescent probe, TAP-4PH, a 1,3a,6a-triazapentalene derivative. TAP-4PH showed high fluorescence in cytoplasmic area, and visualized cytoplasmic and nuclear morphological changes of live cells during differentiation. We demonstrated that TAP-4PH visualized dendritic axon and spine formation in neuronal differentiation, and nuclear structural changes during neutrophilic differentiation. We also showed that the utility of TAP-4PH for visualization of cytoplasmic and nuclear morphologies of various type of live cells. Our visualizing method has no toxicity and no influence on the cellular differentiation and function. The cell morphology can be rapidly observed after addition of TAP-4PH and can continue to be observed in the presence of TAP-4PH in cell culture medium. Moreover, TAP-4PH can be easily removed after observation by washing for subsequent biological assay. Taken together, these results demonstrate that our visualization method is a powerful tool to probe differentiation processes before subsequent biological assay in live cells.

Mutation in lamin A/C sensitizes the myocardium to exercise-induced mechanical stress but has no effect on skeletal muscles in mouse

LMNA gene encodes lamin A/C, ubiquitous proteins of the nuclear envelope. They play crucial role in maintaining nuclear shape and stiffness. When mutated, they essentially lead to dilated cardiomyopathy with conduction defects, associated or not with muscular diseases. Excessive mechanical stress sensitivity has been involved in the pathophysiology. We have previously reported the phenotype of LmnadelK32 mice, reproducing a mutation found in LMNA-related congenital muscular dystrophy patients. Heterozygous LmnadelK32/+ (Het) mice develop a progressive dilated cardiomyopathy leading to death between 35 and 70 weeks of age. To investigate the sensitivity of the skeletal muscles and myocardium to chronic exercise-induced stress, Het and wild-type (Wt) mice were subjected to strenuous running treadmill exercise for 5 weeks. Before exercise, the cardiac function of Het mice was similar to Wt-littermates. After the exercise-period, Het mice showed cardiac dysfunction and dilation without visible changes in cardiac morphology, molecular remodelling or nuclear structure compared to Wt exercised and Het sedentary mice. Contrary to myocardium, skeletal muscle ex vivo contractile function remained unaffected in Het exercised mice. In conclusion, the expression of the LmnadelK32 mutation increased the susceptibility of the myocardium to cardiac stress and led to an earlier onset of the cardiac phenotype in Het mice.

Lateralized Resting-State Functional Brain Network Organization Changes in Heart Failure.

Heart failure (HF) patients show brain injury in autonomic, affective, and cognitive sites, which can change resting-state functional connectivity (FC), potentially altering overall functional brain network organization. However, the status of such connectivity or functional organization is unknown in HF. Determination of that status was the aim here, and we examined region-to-region FC and brain network topological properties across the whole-brain in 27 HF patients compared to 53 controls with resting-state functional MRI procedures. Decreased FC in HF appeared between the caudate and cerebellar regions, olfactory and cerebellar sites, vermis and medial frontal regions, and precentral gyri and cerebellar areas. However, increased FC emerged between the middle frontal gyrus and sensorimotor areas, superior parietal gyrus and orbito/medial frontal regions, inferior temporal gyrus and lingual gyrus/cerebellar lobe/pallidum, fusiform gyrus and superior orbitofrontal gyrus and cerebellar sites, and within vermis and cerebellar areas; these connections were largely in the right hemisphere (p<0.005; 10,000 permutations). The topology of functional integration and specialized characteristics in HF are significantly changed in regions showing altered FC, an outcome which would interfere with brain network organization (p<0.05; 10,000 permutations). Brain dysfunction in HF extends to resting conditions, and autonomic, cognitive, and affective deficits may stem from altered FC and brain network organization that may contribute to higher morbidity and mortality in the condition. Our findings likely result from the prominent axonal and nuclear structural changes reported earlier in HF; protecting neural tissue may improve FC integrity, and thus, increase quality of life and reduce morbidity and mortality.

Disrupted functional brain network organization in patients with obstructive sleep apnea.

IntroductionObstructive sleep apnea (OSA) subjects show impaired autonomic, affective, executive, sensorimotor, and cognitive functions. Brain injury in OSA subjects appears in multiple sites regulating these functions, but the integrity of functional networks within the regulatory sites remains unclear. Our aim was to examine the functional interactions and the complex network organization of these interactions across the whole brain in OSA, using regional functional connectivity (FC) and brain network topological properties.MethodsWe collected resting‐state functional magnetic resonance imaging (MRI) data, using a 3.0‐Tesla MRI scanner, from 69 newly diagnosed, treatment‐naïve, moderate‐to‐severe OSA (age, 48.3 ± 9.2 years; body mass index, 31 ± 6.2 kg/m2; apnea–hypopnea index (AHI), 35.6 ± 23.3 events/h) and 82 control subjects (47.6 ± 9.1 years; body mass index, 25.1 ± 3.5 kg/m2). Data were analyzed to examine FC in OSA over controls as interregional correlations and brain network topological properties.ResultsObstructive sleep apnea subjects showed significantly altered FC in the cerebellar, frontal, parietal, temporal, occipital, limbic, and basal ganglia regions (FDR, P < 0.05). Entire functional brain networks in OSA subjects showed significantly less efficient integration, and their regional topological properties of functional integration and specialization characteristics also showed declined trends in areas showing altered FC, an outcome which would interfere with brain network organization (P < 0.05; 10,000 permutations). Brain sites with abnormal topological properties in OSA showed significant relationships with AHI scores.ConclusionsOur findings suggest that the dysfunction extends to resting conditions, and the altered FC and impaired network organization may underlie the impaired responses in autonomic, cognitive, and sensorimotor functions. The outcomes likely result from the prominent structural changes in both axons and nuclear structures, which occur in the condition.

Abstract A25: A novel spectroscopic technology to image the native chromatin nanostructure in live cells

Abstract Proper regulation of higher-order chromatin structure is essential for normal gene regulation and cellular function. We have previously found that the nanoscale chromatin structure is significantly altered in early and field carcinogenesis using novel spectroscopic methods in parallel with biological assays (Backman and Roy, J Cancer, 2013, 3:251-261; Subramanian et al, Cancer Res, 2009, 13:5357-63; Stypula-Cyrus et al, PLoS One, 2013, 5:e64600). This was done in fixed human and animal model samples, suggesting that genetic/epigenetic alterations can serve as the earliest marker for neoplastic transformation. While chromatin is well understood at the nucleosomal level (&amp;lt;20nm) and chromosomal level (&amp;gt;200nm), little is known about the higher-order chromatin structure between these length scales. Current techniques available to study cellular structures below the diffraction limit (&amp;lt;200nm) require labeling that may alter the native cell structure, can only visualize a few molecules concurrently, and have poor temporal resolution. Here we present a new technique, BAck-Scattering Interference Spectroscopic (BaSIS) microscopy, with sensitivity to structures between 20-200nm that can quantify the dynamics of the nano-molecular organization in live cells without using exogenous labels. The BaSIS instrument was built into a commercial inverted microscope (Leica DMIRB) equipped with a high NA oil immersion objective with broadband illumination provided by a Xenon lamp. Refractive index fluctuations are measured by sampling backscattered light at each wavelength 500-700nm using a combination of a liquid crystal tunable filter (LCTF) and a CMOS camera. HeLa and CHO cells were first imaged in petri dishes with coverslip bottoms, and then incubated with Hoechst 33342, a nuclear stain that binds to AT-rich regions of the genome and has been reported to cause double-stranded breaks (DSBs) in the DNA (Pfeiffer et al, Mutagenesis, 2000, 4: 289-302). Additionally, mock-staining experiments were performed to compare the changes in nuclear structure due to Hoechst 33342 excitation compared to UV light exposure alone. We utilized a γ-H2A.X-Alexa488 conjugated antibody after Hoechst- and mock-staining to compare observed changes in BaSIS signal with the formation of DSBs. Using BaSIS, we show for the first time that the excitation of Hoechst 33342 immediately alters the native nuclear nanostructure and induces formation of DSBs, confirmed by the rapid phosphorylation of H2A.X. In our mock-stained control, we observed an average increase of 0.006% and 0.001% signal after UV exposure (p-value &amp;gt; 0.5), whereas the stained cells display a 17.01% and 7.1% decrease in HeLa and CHO nuclei, respectively (p-value &amp;lt; 0.001). Significantly, these changes in Hoechst-stained cells are detectable by BaSIS within seconds. This suggests that the mechanism responsible for the nuclear alteration detected by BaSIS is localized and immediate. We hypothesized that the observed transformation was due to the homogenization of chromatin through fragmentation and the concurrent chromatin decompaction, detected by refractive index fluctuations. Indeed, following Hoechst staining, we observed an accumulation of the γ-H2A.X antibody, whereas we observed little or no localization in the mock Hoechst-stained nuclei. Furthermore, traditional phase contrast microscopy, which is used for label-free imaging of living samples, did not show any changes in the higher-order chromatin structure following DSB formation in these experiments. In conclusion, BaSIS is a powerful tool for studying the dynamics of chromatin nanostructure and can serve as a natural supplement to super-resolution fluorescence techniques, providing quantified information about native cellular organization. With this technique, we demonstrated that using the Hoechst DNA-binding dye causes irreversible alterations in chromatin structure at time-scales (seconds) not previously recognized. As a result, BASIS can be applied to a broad range of critical studies in chromatin research. Current and future research include: (i) mRNA transport and the accessibility of euchromatin and heterochromatin to transcription factors; (ii) why and how high-order chromatin structure changes in cancer progression; (iii) the role of nuclear architecture as an epigenetic regulator of gene expression; and (iv) the effect of metabolism on chromatin structure; (v) damage/repair mechanisms and potentially, chemotherapeutic efficacy. Citation Format: Yolanda Stypula, Scott Gladstein, Luay Almassalha, Greta Bauer, John Chandler, Lusik Cherkezyan, Di Zhang, Hariharan Subramanian, Igal Szleifer, Vadim Backman. A novel spectroscopic technology to image the native chromatin nanostructure in live cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A25.

Conformational dynamics of stem II of the U2 snRNA.

The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg2+ and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg2+ and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg2+ may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg2+ or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg2+ toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg2+-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.

Abstract 12095: Altered Brain Functional Connectivity During Valsalva Maneuver in Heart Failure

Introduction: Heart failure (HF) patients have abnormal autonomic (ANS) activity, which is linked to damage in specific gray matter brain regions (such as the insulae). Abnormal ANS activity in HF may be due to changes in both structural injury and functional connectivity (FC; temporal correlations between neural structures which indicate connection status and sequence of activation). While insular/brain structural injury has been reported in HF, FC has not been explored. Hypothesis: FC between brain regions during an autonomic challenge will be less in HF in comparison to controls. Methods: We collected functional MRI data with Valsalva maneuver block design task in 12 HF (age 55.6±8.1 years; Females 3; BMI 25.2±3.8 kg/m2; LVEF, 26.7±11.5%) and 33 controls (51.6±5.5 years; Females 13; BMI 25.7±3.2 kg/m2), using a 3.0 Tesla MRI scanner. Group independent component analysis was applied to identify individual task-related FC maps, which were compared between HF and controls (ANCOVA; covariates, age and gender; P&lt;0.005) Results: No significant differences in age, gender, or BMI emerged between the two groups (P&gt;0.05). Decreased FC in HF (Figure 1; p&lt;0.005) were found in the left supramarginal gyrus/inferior parietal lobule, left middle temporal gyrus, bilateral thalamus, left fusiform gyrus, left ligual gyrus, right precuneus, left hippocampus, and left insula. However, increased FC in HF compared to controls were found in the left inferior frontal gyrus, right superior temporal gyrus, left cerebellar cortex, and right insula (P&lt;0.005). Conclusions: These findings suggest that autonomic, cognitive, and affective deficits in HF may be related to altered FC. The outcomes likely result from the prominent structural changes in both axons and nuclear structures reported earlier in HF. Future interventions may need to address structural and functional deficits in both gray and white matter regions in HF.

Reduced transition strengths of low-lying yrast states in chromium isotopes in the vicinity ofN=40

Background: In neutron-rich nuclei around $N=40$ rapid changes in nuclear structure can be observed. While $^{68}\mathrm{Ni}$ exhibits signatures of a doubly magic nucleus, experimental data along the isotopic chains in even more exotic Fe and Cr isotopes---such as excitation energies and transition strengths---suggest a sudden rise in collectivity toward $N=40$.Purpose: Reduced quadrupole transition strengths for low-lying transitions in neutron-rich $^{58,60,62}\mathrm{Cr}$ are investigated. This gives quantitative new insights into the evolution of quadrupole collectivity in the neutron-rich region close to $N=40$.Method: The recoil distance Doppler-shift (RDDS) technique was applied to measure lifetimes of low-lying states in $^{58,60,62}\mathrm{Cr}$. The experiment was carried out at the National Superconducting Cyclotron Laboratory (NSCL) with the SeGA array in a plunger configuration coupled to the S800 magnetic spectrograph. The states of interest were populated by means of one-proton knockout reactions.Results: Data reveal a rapid increase in quadrupole collectivity for $^{58,60,62}\mathrm{Cr}$ toward $N=40$ and point to stronger quadrupole deformations compared to neighboring Fe isotopes. The experimental $B(E2$) values are reproduced well with state-of-the-art shell-model calculations using the LNPS effective interaction. A consideration of intrinsic quadrupole moments and ${\mathrm{B}}_{42}$ ratios suggest an evolution toward a rotational nature of the collective structures in $^{60,62}\mathrm{Cr}$. Compared to $^{58}\mathrm{Cr}$, experimental ${\mathrm{B}}_{42}$ and ${\mathrm{B}}_{62}$ values for $^{60}\mathrm{Cr}$ are in better agreement with the $E(5)$ limit.Conclusion: Our results indicate that collective excitations in neutron-rich Cr isotopes saturate at $N=38$, which is in agreement with theoretical predictions. More detailed experimental data of excited structures and interband transitions are needed for a comprehensive understanding of quadrupole collectivity close to $N=40$. This calls for additional measurements in neutron-rich Cr and neighboring Ti and Fe nuclei.

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.

Nuclear Actin Dynamics Regulate Nuclear Organization and Transcription

2716-Pos Board B146 Nuclear Actin Dynamics Regulate Nuclear Organization and Transcription Leo Serebryannyy 1 , Megan Parilla 1 , Paolo Annibale 2 , Christina Cruz 1 , Dmitri Kudryashov 3 , Enrico Gratton 2 , Cara J. Gottardi 4 , Primal de Lanerolle 1 . Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA, 2 Laboratory of Fluorescence Dynamics, University of California at Irvine, Irvine, CA, USA, 3 Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, 4 Medicine, Northwestern University, Chicago, IL, USA. Actin is an important and elegant mechanotransducer that transfers messages from the extracellular environment into the nucleus, thus conferring changes in both the physical properties and the genetic program of the cell. Although actin in the nucleus has been established in a growing number of functions, the form of nuclear actin remains poorly understood as there are no apparent actin filaments in the nucleus. Therefore, we tested how disrupting the form of nuclear actin impacts function and what effects this may have on the nucleus. To do so, we formed stable nuclear actin filaments using a variety of methods, including the nuclear enrichment of actin binding proteins supervillin and a-E-catenin and the expression of a mutant form of skeletal a-actin. Using fluorescence recovery after photobleaching (FRAP), we found that stabiliza- tion of nuclear actin filaments significantly impairs actin dynamics within the nucleus. The formation of nuclear actin filaments coincides with striking changes in nuclear structure and overall nuclear topography as determined by confocal microscopy and raster image correlation spectroscopy. Using a com- bination of FRAP analysis, structured illumination microscopy and immuno- logical assays, we found that nuclear actin filaments reduce the association of actin with RNA polymerase II and this correlates with impaired RNA poly- merase II dynamics, localization and gene recruitment. Moreover, we were able to recapitulate our findings in purified nuclear extract by using in vitro transcription assays with the covalent actin crosslinking domain (ACD) of V. cholerae MARTX toxin. Based on our data, which help explain the absence of nuclear actin filaments in the interphase mammalian nucleus, we propose a model where nuclear actin dynamics are critical for maintaining proper nu- clear function.

Epithelial-mesenchymal transition in prostate cancer is associated with quantifiable changes in nuclear structure.

Prostate cancer progression is concomitant with quantifiable nuclear structure and texture changes as compared to non-cancer tissue. Malignant progression is associated with an epithelial-mesenchymal transition (EMT) program whereby epithelial cancer cells take on a mesenchymal phenotype and dissociate from a tumor mass, invade, and disseminate to distant metastatic sites. The objective of this study was to determine if epithelial and mesenchymal prostate cancer cells have different nuclear morphology. Murine tibia injections of epithelial PC3 (PC3-Epi) and mesenchymal PC3 (PC3-EMT) prostate cancer cells were processed and stained with H&E. Cancer cell nuclear image data was obtained using commercially available image-processing software. Univariate and multivariate statistical analysis were used to compare the two phenotypes. Several non-parametric classifiers were constructed and permutation-tested at various training set fractions to ensure robustness of classification between PC3-Epi and PC3-EMT cells in vivo. PC3-Epi and PC3-EMT prostate cancer cells were separable at the single cell level in murine tibia injections on the basis of nuclear structure and texture remodeling associated with an EMT. Support vector machine and multinomial logistic regression models based on nuclear architecture features yielded AUC-ROC curves of 0.95 and 0.96, respectively, in separating PC3-Epi and PC3-EMT prostate cancer cells in vivo. Prostate cancer cells that have undergone an EMT demonstrated an altered nuclear structure. The association of nuclear changes and a mesenchymal phenotype demonstrates quantitative morphometric image analysis may be used to detect cancer cells that have undergone EMT. This morphometric measurement could provide valuable prognostic information in patients regarding the likelihood of [future] metastatic disease.