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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
NASA NTRS2001

Use of abnormal preprophase bands to decipher division plane determination

Many premitotic plant cells possess a cortical preprophase band of microtubules and actin filaments that encircles the nucleus. In vacuolated cells, the preprophase band is visibly connected to the nucleus by a cytoplasmic raft of actin filaments and microtubules termed the phragmosome. Typically, the location of the preprophase band and phragmosome corresponds to, and thus is thought to influence, the location of the cell division plane. To better understand the function of the preprophase band and phragmosome in orienting division, we used a green fluorescent protein-based microtubule reporter protein to observe mitosis in living tobacco bright yellow 2 cells possessing unusual preprophase bands. Observations of mitosis in these unusual cells support the involvement of the preprophase band/phragmosome in properly positioning the preprophase nucleus, influencing spindle orientation such that the cytokinetic phragmoplast initially grows in an appropriate direction, and delineating a region in the cell cortex that attracts microtubules and directs later stages of phragmoplast growth. Thus, the preprophase band/phragmosome appears to perform several interrelated functions to orient the division plane. However, functional information associated with the preprophase band is not always used or needed and there appears to be an age or distance-dependent character to the information. Cells treated with the anti-actin drug, latrunculin B, are still able to position the preprophase nucleus suggesting that microtubules may play a dominant role in premitotic positioning. Furthermore, in treated cells, spindle location and phragmoplast insertion are frequently abnormal suggesting that actin plays a significant role in nuclear anchoring and phragmoplast guidance. Thus, the microtubule and actin components of the preprophase band/phragmosome execute complementary activities to ensure proper orientation of the division plane.

NASA Discipline Plant Biology↗
NASA NTRS1995

Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS).

NASA Discipline Cell Biology↗
DOE OSTI2026

Localization and functional exploration of leiomodin-2’s C-terminal binding sites

Striated muscle contraction occurs through interactions between overlapping myosin-based thick and actin-based thin filaments within the sarcomere. For effective contraction to occur, the length of the thin filament must be maintained to allow for sufficient overlap with the thick filament. The proteins leiomodin and tropomodulin compete for binding at the pointed end of thin filaments to regulate their length, utilizing their homologous N-terminal actin and tropomyosin binding sites. Leiomodin also has a region called the C-terminal extension, absent in tropomodulin. In this region, the cardiac isoform (leiomodin-2) contains additional actin-binding sites that enable it to bind along the sides of thin filaments in a Ca2+-dependent manner. Here, using nuclear magnetic resonance spectroscopy, we localize the regions of the C-terminal extension that contain residues involved in thin filament side-binding. Using co-sedimentation assays, we reveal that these regions can independently bind thin filaments and discover that the poly-proline region plays a role as a linker, maintaining an adequate distance between two of the regions required for effective interaction to occur. In addition to its role in side-binding, we provide direct evidence that the poly-proline region interacts with profilin and propose a new mechanism by which leiomodin-2 may assist in the polymerization of profilin-bound actin at thin filament pointed ends.

ACTIN-BINDING PROTEINS↗
DOE OSTI2017

The reason for a high Ca2+-sensitivity associated with Arg91Gly substitution in TPM2 gene is the abnormal behavior and high flexibility of tropomyosin during the ATPase cycle

Highlights: • Mutation R91G initiates abnormally high flexibility of β-tropomyosin (TM). • Troponin moves R91G-TM to the blocked position at low Ca{sup 2+}. • This mutation does not alter the ability of troponin to switch off actin monomers. • R91G-TM is located near the open position during the ATPase cycle at low Ca{sup 2+}. • Mutation R91G increases the fraction of rigor myosin heads at low Ca{sup 2+}. Substitution of Arg for Gly residue in 91th position in β-tropomyosin caused by a point mutation in TPM2 gene is associated with distal arthrogryposis, characterized by a high Ca{sup 2+}-sensitivity of myofilament and contracture syndrome. To understand the mechanisms of this defect, we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres, using the polarized fluorescence microscopy. The mutation was shown to markedly decrease the bending stiffness of β-tropomyosin in the thin filaments. In the absence of the myosin heads the mutation did not alter the ability of troponin to shift tropomyosin to the blocked position and to switch actin monomers off at low Ca{sup 2+}. During the ATPase cycle the movement of the mutant tropomyosin is restrained, it is located near the open position, which allows strong binding of the myosin heads to actin even at low Ca{sup 2+}. This may be the reason for both high Ca{sup 2+}-sensitivity and contractures associated with the Arg91Gly mutation. The use of reagents that decrease the Ca{sup 2+}sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PFN2a, a new partner of RARα in the cytoplasm

Highlights: • RARα, though mainly a nuclear transcription factor, is also present in the cytoplasm. • In the cytoplasm, RARα interacts with PFN2a, a small G-actin-binding protein. • When increased in the cytoplasm, RARα impedes microfilaments elongation. Retinoic acid receptors (RARs) are classically considered as nuclear ligand-dependent regulators of transcription. Here we highlighted a novel face of the RARα subtype: RARα is present in low amounts in the cytoplasm of mouse embryonic fibroblasts (MEFs) where it interacts with profilin2a (PFN2A), a small actin-binding protein involved in filaments polymerization. The interaction involves the N-terminal proline-rich motif (PRM) of RARα and the SH3-like domain of PFN2a. When increased in the cytoplasm, RARα competes with other PFN2a-binding proteins bearing PRMs and involved in actin filaments elongation. Consequently, the actin filament network is altered and MEFs adhesion is decreased. This novel role opens novel avenues for the understanding of pathologies characterized by increased levels of cytoplasmic RARα.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Cullin 3 regulates ADAMs-mediated ectodomain shedding of amphiregulin

Highlights: • ADAMs-mediated EGF family shedding is regulated by actin cytoskeleton. • Cul3 regulates AREG shedding by modulating actin cytoskeleton. • The neddylation is crucial step for Cul3 to regulate ectodomain shedding. • Cul3 E3 ubiquitin ligase complex is promising target to regulate ADAM activity. A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Characterization of SETD3 methyltransferase–mediated protein methionine methylation

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these cases, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain–containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr 312 and Trp 273 , as well as the hydrocarbon side chain of Ile 310 . Our results suggest that placing methionine properly in the active site—within close proximity to and in line with the incoming methyl group of SAM—would allow some SET domain proteins to selectively methylate methionine in proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion

Dynamic communication between integrin-containing complexes (focal adhesions, FAs) and actin filaments is critical for regulating cell adhesion. Pseudokinase ILK plays a key role in this process but the underlying mechanism remains highly elusive. Here in this paper we show that by recruiting FA adaptors PINCH and Parvin into a heterotrimeric complex (IPP), ILK triggers F-actin filament bundling – a process known to generate force/mechanical signal to promote cytoskeleton reassembly and dynamic cell adhesion. Structural, biochemical, and functional analyses revealed that the F-actin bundling is orchestrated by two previously unrecognized WASP-Homology-2 actin binding motifs within IPP, one from PINCH and the other from Parvin. Strikingly, this process is also sensitized to Mg-ATP bound to the pseudoactive site of ILK and its dysregulation severely impairs stress fibers formation, cell spreading, and migration. These data identify a crucial mechanism for ILK, highlighting its uniqueness as a pseudokinase to transduce non-catalytic signal and regulate cell adhesion.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity

Various mutations in the structural proteins nebulin and titin that are present in human disease are known to affect the contractility of striated muscle. Loss of nebulin is associated with reduced actin filament length and impairment of myosin binding to actin, whereas titin is thought to regulate muscle passive elasticity and is likely involved in length-dependent activation. Here, we sought to assess the modulation of muscle function by these sarcomeric proteins by using the computational platform muscle simulation code (MUSICO) to quantitatively separate the effects of structural changes, kinetics of cross-bridge cycling, and calcium sensitivity of the thin filaments. The simulations show that variation in thin filament length cannot by itself account for experimental observations of the contractility in nebulin-deficient muscle, but instead must be accompanied by a decreased myosin binding rate. Additionally, to match the observed calcium sensitivity, the rate of TnI detachment from actin needed to be increased. Simulations for cardiac muscle provided quantitative estimates of the effects of different titin-based passive elasticities on muscle force and activation in response to changes in sarcomere length and interfilament lattice spacing. Predicted force–pCa relations showed a decrease in both active tension and sensitivity to calcium with a decrease in passive tension and sarcomere length. We conclude that this behavior is caused by partial redistribution of the muscle load between active muscle force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, which together modulate the release of TnI from actin. These data help advance understanding of how nebulin and titin mutations affect muscle function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Myosin dilated cardiomyopathy mutation S532P disrupts actomyosin interactions, leading to altered muscle kinetics, reduced locomotion, and cardiac dilation in Drosophila

Dilated cardiomyopathy (DCM), a life-threatening disease characterized by pathological heart enlargement, can be caused by myosin mutations that reduce contractile function. To better define the mechanistic basis of this disease, we employed the powerful genetic and integrative approaches available in Drosophila melanogaster. To this end, we generated and analyzed the first fly model of human myosin–induced DCM. The model reproduces the S532P human β-cardiac myosin heavy chain DCM mutation, which is located within an actin-binding region of the motor domain. In concordance with the mutation’s location at the actomyosin interface, steady-state ATPase and muscle mechanics experiments revealed that the S532P mutation reduces the rates of actin-dependent ATPase activity and actin binding and increases the rate of actin detachment. The depressed function of this myosin form reduces the number of cross-bridges during active wing beating, the power output of indirect flight muscles, and flight ability. Further, S532P mutant hearts exhibit cardiac dilation that is mutant gene dose–dependent. Our study shows that Drosophila can faithfully model various aspects of human DCM phenotypes and suggests that impaired actomyosin interactions in S532P myosin induce contractile deficits that trigger the disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. For this study, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Quantum-enhanced photoprotection in neuroprotein architectures emerges from collective light-matter interactions

Background Superradiance is the phenomenon of many identical quantum systems absorbing and/or emitting photons collectively at a higher rate than any one system can individually. This phenomenon has been studied analytically in idealized distributions of electronic two-level systems (TLSs), each with a ground and excited state, as well as numerically in realistic photosynthetic nanotubes and cytoskeletal architectures. Methods Superradiant effects are studied here in idealized toy model systems and realistic biological mega-networks of tryptophan (Trp) molecules, which are strongly fluorescent amino acids found in many proteins. Each Trp molecule acts as a chromophore absorbing in the ultraviolet spectrum and can be treated approximately as a TLS, with its 1 L a excited singlet state; thus, organized Trp networks can exhibit superradiance. Such networks are found, for example, in microtubules, actin filaments, and amyloid fibrils. Microtubules and actin filaments are spiral-cylindrical protein polymers that play significant biological roles as primary constituents of the eukaryotic cytoskeleton, while amyloid fibrils have been targeted in a variety of neurodegenerative diseases. We treat these proteinaceous Trp networks as open quantum systems, using a non-Hermitian Hamiltonian to describe interactions of the chromophore network with the electromagnetic field. We numerically diagonalize the Hamiltonian to obtain its complex eigenvalues, where the real part is the energy and the imaginary part is its associated enhancement rate. We also consider multiple realizations of increasing static disorder in either the site energies or the decay rates. Results We obtained the energies and enhancement rates for realistic microtubules, actin filament bundles, and amyloid fibrils of differing lengths, and we use these values to calculate the quantum yield, which is the ratio of the number of photons emitted to the number of photons absorbed. We find that all three of these structures exhibit highly superradiant states near the low-energy portion of the spectrum, which enhances the magnitude and robustness of the quantum yield to static disorder and thermal noise. Conclusion The high quantum yield and stable superradiant states in these biological architectures may play a photoprotective rolein vivo, downconverting energetic ultraviolet photons—absorbed from those emitted by reactive free radical species—to longer, safer wavelengths and thereby mitigating biochemical stress and photophysical damage. Contrary to conventional assumptions that quantum effects cannot survive in large biosystems at high temperatures, our results suggest that macropolymeric collectives of TLSs in microtubules, actin filaments, and amyloid fibrils exhibit increasingly observable and robust effects with increasing length, up to the micron scale, due to quantum coherent interactions in the single-photon limit. Superradiant enhancement and high quantum yield exhibited in neuroprotein polymers could thus play a crucial role in information processing in the brain, the development of neurodegenerative diseases such as Alzheimer’s and related dementias, and a wide array of other pathologies characterized by anomalous protein aggregates.

Physics↗
NASA NTRS1999

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

Rosenthal, Nadia A.↗
NASA NTRS2001

Hypergravity Loading the Cultured Osteoblasts: Modeling and Experimental Analysis of Cellular Morphology and the Cytoskeleton

Bone forming cells, osteoblasts, respond to various mechanical forces, including mechanical strain and fluid-induced shear stress. This study examined whether osteoblasts detect changes in gravity as a mechanical force, as assessed by cellular morphology and dimensions of the cytoskeletal network. We used modeling to evaluate how gravity influences cell morphology given theoretical differences in densities between the surrounding medium, cytoplasm, and nucleus. A mechanical model was built based on analysis of axisymmetric shell structures (Fast4 software) to study the effects of 10 times gravity (10G) on cell height. The model indicated 0.02% decrease in overall cell height when the medium was 10% denser than the nucleus or cytoplasm, 5.9 x 10(exp-5)% decrease when the nucleus was 10% denser than the cytoplasm or medium, and 1.3 x 10(exp-5)% decrease when the cell cytoplasm was 10% denser than the nucleus or medium. To experimentally evaluate the influence of gravity, cultured primary fetal rat osteoblasts were grown to near confluence and centrifuged at 10G for 3 hours. Actin, microtubules, and nuclei were fluorescently labeled and analyzed by confocal microscopy to determine overall microtubule and actin network height. Centrifugation led to an apparent reduction in height of both the microtubule (-16%) and the actin (-20%) networks relative to stationary controls. Thus, both modeling and experiments indicate that hypergravity reduces the height of the osteoblast cell layer and their microtubule and actin networks. This combination of modeling and experimental analyses will help us to better understand the mechanical loading of osteoblasts.

Searby, N. D.↗
NASA NTRS2002

Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation

Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.

Non-NASA Center↗
DOE OSTI2016

In vivo epidermal migration requires focal adhesion targeting of ACF7

Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7's NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo . Altogether, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Up-regulation of GTPBP4 in colorectal carcinoma is responsible for tumor metastasis

GTP binding protein 4(GTPBP4), a member of GTP-binding protein family, was previously characterized as a tumor suppressor that regulates and requires merlin to suppress cell proliferation. However, the role of GTPBP4 in the metastasis of colorectal carcinoma (CRC) remains unelucidated. Here, we observed that GTPBP4 was detected at higher levels in CRC metastatic tissues than that in the primary tumor tissues. Notably, up-regulation of GTPBP4 was closely correlated with tumor metastasis in CRCs. Kaplan-Meier and multivariate Cox regression analysis indicated GTPBP4 as an independent prognostic factor for CRC patients (hazard ratio = 2.693, 95% confident interval: 1.193–6.083, p = 0.017). Functional studies established that knockdown of GTPBP4 impeded, whereas ectopic expression of GTPBP4 enhanced cell motility and tumor metastasis in CRC cells. Interestingly, mechanistic investigations suggested that GTPBP4 may disorganize actin cytoskeleton through repressing RhoA signaling. Taken together, our research uncovered that GTPBP4 promotes CRC metastasis by disrupting actin cytoskeleton, which is mediated by the reduced RhoA activity. Strategies targeting GTPBP4 will be promising for CRC patients with metastases. - Highlights: • Up-regulation of GTPBP4 is detected in CRC metastatic tissues and closely correlated with tumor metastasis. • Increase of GTPBP4 is closely associated with poor prognosis. • GTPBP4 promotes cell motility and tumor metastasis in CRC cells. • GTPBP4 induces filamentous actin rearrangement specifically by repressing the activity of RhoA. • GTPBP4 may be a novel therapeutic target for CRC patients with metastasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Cell membrane dynamics induction using optogenetic tools

Highlights: • Actin-based cell membrane movements play important roles in various cellular functions. • Recently, we developed a brand-new optogenetic tool, called the Magnet system. • This tool enabled us to spatiotemporally induce the change in cell membrane dynamics. • In this review, we summarized recent advances in optogenetic tools and our Magnet system. Structures arising from actin-based cell membrane movements, including ruffles, lamellipodia, and filopodia, play important roles in a broad spectrum of cellular functions, such as cell motility, axon guidance in neurons, wound healing, and micropinocytosis. Previous studies investigating these cell membrane dynamics often relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies did not allow the modulation of protein activity at specific regions of cells, tissues, and organs in animals with high spatial and temporal precision. Recently, optogenetic tools for inducing cell membrane dynamics have been developed which address several disadvantages of previous techniques. In a recent study, we developed a powerful optogenetic tool, called the Magnet system, to change cell membrane dynamics through Tiam1 and PIP3 signal transductions with high spatial and temporal resolution. In this review, we summarize recent advances in optogenetic tools that allow us to induce actin-regulated cell membrane dynamics and unique membrane ruffles that we discovered using our Magnet system.

60 APPLIED LIFE SCIENCES↗