Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core ...FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIP
into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.
Controlling cell functions using external photoresponsive nanomaterials has enormous potential for the development of cell‐engineering technologies and intractable disease therapies, but the former ...currently requires genetic modification of the target cells. We present a method using plasma‐membrane‐targeted gold nanorods (pm‐AuNRs) prepared with a cationic protein/lipid complex to activate a thermosensitive cation channel, TRPV1, in intact neuronal cells. Highly localized photothermal heat generation mediated by the pm‐AuNRs induced Ca2+ influx solely by TRPV1 activation. In contrast, the use of previously reported cationic AuNRs that are coated with a conventional synthetic polymer also led to photoinduced Ca2+ influx, but this influx resulted from membrane damage. Our method provides an optogenetic platform without the need for prior genetic engineering of the target cells and might be useful for novel TRPV1‐targeted phototherapeutic approaches.
Hot membranes: A non‐disruptive plasma membrane heating method employs gold nanorods (AuNRs) coated with a cationic protein/lipid complex. Under near‐infrared illumination, these AuNRs induce highly localized photothermal heat generation in intact neuronal cells without membrane damage, enabling Ca2+ influx solely by activation of the thermosensitive cation channel TRPV1.
The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in ...membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.
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► Acute osmotic swelling or cell stretching induces rapid caveola flattening and disassembly ► Caveola disassembly is mechanically driven in an actin- and ATP-independent manner ► Stress relaxation leads to caveola reassembly in an ATP-dependent manner ► Caveola flattening buffers membrane tension variations and prevents membrane rupture
Porous coordination polymers (PCPs) are an intriguing class of molecular-based materials because of the designability of framework scaffolds, pore sizes and pore surface functionalities. Besides the ...structural designability at the molecular scale, the structuring of PCPs into mesoscopic/macroscopic morphologies has attracted much attention due to the significance for the practical applications. The structuring of PCPs at the mesoscopic/macroscopic scale has been so far demonstrated by the spatial localization of coordination reactions on the surface of templates or at the phase boundaries. However, these methodologies have never been applied to the fabrication of solid-solution or multivariate metal–organic frameworks (MOFs), in which multiple components are homogeneously mixed. Herein, we demonstrate the structuring of a box-type superstructure comprising of a solid-solution PCP by integrating a bidirectional diffusion of multiple organic ligands into molecular assembly. The parent crystals of Zn2(ndc)2(bpy) n were placed in the DMF solution of additional organic component of H2bdc, and the temperature was rapidly elevated up to 80 °C (ndc = 1,4-naphthalenedicarboxylate, bpy = 4,4′-bipyridyl, bdc = 1,4-benzenedicarboxylate). The dissolution of the parent crystals induced the outward diffusion of components; contrariwise, the accumulation of the other organic ligand of H2bdc induced the inward diffusion toward the surface of the parent crystals. This bidirectional diffusion of multiple components spatially localized the recrystallization at the surface of cuboid parent crystals; therefore, the nanocrystals of a solid-solution PCP (Zn2(bdc)1.5(ndc)0.5(bpy) n ) were organized into a mesoscopic box superstructure. Furthermore, we demonstrated that the box superstructures enhanced the mass transfer kinetics for the separation of hydrocarbons.
The transcriptional corepressor complex CoREST is one of seven histone deacetylase complexes that regulate the genome through controlling chromatin acetylation. The CoREST complex is unique in ...containing both histone demethylase and deacetylase enzymes, LSD1 and HDAC1, held together by the RCOR1 scaffold protein. To date, it has been assumed that the enzymes function independently within the complex. Now, we report the assembly of the ternary complex. Using both structural and functional studies, we show that the activity of the two enzymes is closely coupled and that the complex can exist in at least two distinct states with different kinetics. Electron microscopy of the complex reveals a bi-lobed structure with LSD1 and HDAC1 enzymes at opposite ends of the complex. The structure of CoREST in complex with a nucleosome reveals a mode of chromatin engagement that contrasts with previous models.
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•The activities of LSD1 and HDAC1 are closely coupled in the CoREST complex•Both LSD1 and HDAC1 exist in two different kinetic states•CoREST has a bi-lobed, flexible structure with the two enzymes located at opposite ends•CoREST interacts with methylated nucleosomes via LSD1, but not HDAC1 or RCOR1
Using a real-time NMR assay, Song et al. characterize crosstalk between LSD1 and HDAC1 in the CoREST complex. Activation or inhibition of one enzyme strongly affects activity of the other. Electron microscopy studies of the complex reveal a bi-lobed structure with implications for the mode of interaction with nucleosomes.
Naive pluripotent stem cells (PSCs) utilize both glycolysis and oxidative phosphorylation (OXPHOS) to satisfy their metabolic demands. However, it is unclear how somatic cells acquire this hybrid ...energy metabolism during reprogramming toward naive pluripotency. Here, we show that when transduced with Oct4, Sox2, and Klf4 (OSK) into murine fibroblasts, Zic3 and Esrrb synergistically enhance the reprogramming efficiency by regulating cellular metabolic pathways. These two transcription factors (TFs) cooperatively activate glycolytic metabolism independently of hypoxia inducible factors (HIFs). In contrast, the regulatory modes of the TFs on OXPHOS are antagonistic: Zic3 represses OXPHOS, whereas Esrrb activates it. Therefore, when introduced with Zic3, Esrrb restores OXPHOS activity, which is essential for efficient reprogramming. In addition, Esrrb-mediated OXPHOS activation is critical for the conversion of primed PSCs into the naive state. Our study suggests that the combinatorial function of TFs achieves an appropriate balance of metabolic pathways to induce naive PSCs.
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•Zic3 and Esrrb synergistically enhance somatic cell reprogramming•Zic3 and Esrrb cooperatively bind and activate glycolysis genes•Esrrb activates OXPHOS, but Zic3 represses it•OXPHOS activation enhances reprogramming of EpiSCs
Sone et al. show that the transcription factors Zic3 and Esrrb synergistically enhance the reprogramming efficiency of murine fibroblasts transduced with the classic Oct4, Sox2, and Klf4 cocktail to induce naive pluripotency by regulating cellular metabolic pathways. Zic3 and Esrrb achieve a delicate orchestrated balance of glycolysis and oxidative phosphorylation in PSCs.
A reliable extraction technique for resolving multiple spots in light or electron microscopic images is essential in investigations of the spatial distribution and dynamics of specific proteins ...inside cells and tissues. Currently, automatic spot extraction and characterization in complex microscopic images poses many challenges to conventional image processing methods.
A new method to extract closely located, small target spots from biological images is proposed. This method starts with a simple but practical operation based on the extended morphological top-hat transformation to subtract an uneven background. The core of our novel approach is the following: first, the original image is rotated in an arbitrary direction and each rotated image is opened with a single straight line-segment structuring element. Second, the opened images are unified and then subtracted from the original image. To evaluate these procedures, model images of simulated spots with closely located targets were created and the efficacy of our method was compared to that of conventional morphological filtering methods. The results showed the better performance of our method. The spots of real microscope images can be quantified to confirm that the method is applicable in a given practice.
Our method achieved effective spot extraction under various image conditions, including aggregated target spots, poor signal-to-noise ratio, and large variations in the background intensity. Furthermore, it has no restrictions with respect to the shape of the extracted spots. The features of our method allow its broad application in biological and biomedical image information analysis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Two very polarized views exist for understanding the cellular plasma membrane (PM). For some, it is the simple fluid described by the original Singer-Nicolson fluid mosaic model. For others, due to ...the presence of thousands of molecular species that extensively interact with each other, the PM forms various clusters and domains that are constantly changing and therefore, no simple rules exist that can explain the structure and molecular dynamics of the PM. In this article, we propose that viewing the PM from its two predominant components, cholesterol and actin filaments, provides an excellent and transparent perspective of PM organization, dynamics, and mechanisms for its functions. We focus on the actin-induced membrane compartmentalization and lipid raft domains coexisting in the PM and how they interact with each other to perform PM functions. This view provides an important update of the fluid mosaic model.
To visualize the basal or apical cytoplasmic surface just beneath the plasma membrane, we developed two different methods (“unroof” and “rip-off”). The immunoreplica technique for “unroof” and ...“rip-off” sample preparation that will be presented in this chapter can determine the distributions of actin, actin-binding proteins, transmembrane proteins, and membrane lipids at the interface of the plasma membrane. We have currently developed freeze-etch electron tomography, which could visualize the 3D molecular architecture of membrane-associated structures (membrane skeleton, clathrin-coated pits, and caveolae) on the cytoplasmic surface of the plasma membrane with 0.85-nm-thick consecutive sections made ≈100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeletons that are closely apposed to the plasma membrane interface are suggested to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules.
Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, ...clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.
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► KY02111 promotes the differentiation of hPSCs to functional cardiomyocytes ► KY02111 acts downstream of APC and GSK3β to inhibit WNT signaling ► KY02111 and WNT inhibitors cooperatively enhance hPSC cardiomyogenesis ► KY02111and WNT modulators permit cytokine and xeno-free hPSC cardiomyogenesis
Human pluripotent stem cell (hPSC)-based regenerative therapies for heart disease require clinical-grade cardiac cells produced from hPSCs in a defined, cost-effective manner. Uesugi, Aiba, and colleagues have discovered a small molecule, KY02111, that promotes differentiation of hPSCs to cardiomyocytes by inhibiting WNT signaling. KY02111 and other WNT signaling modulators produce robust cardiac differentiation of hPSCs under cytokine- and xeno-free conditions. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.
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