Class II myosins generate contractile forces in cells by polymerizing into bipolar filaments and pulling on anchored actin filaments. Nonmuscle myosin II (NMII) plays central roles during cell ...adhesion, migration, cytokinesis, and tissue morphogenesis. NMII is present in virtually all mammalian cell types as tissue-specific combinations of NMIIA, NMIIB, and NMIIC isoforms. It remains poorly understood how the highly dynamic NMII-actin contractile system begins to assemble at new cellular locations during cell migration and how incorporation of different NMII isoforms into this system is coordinated.
Using platinum replica electron microscopy in combination with immunogold labeling, we demonstrate that individual activated (phosphorylated on the regulatory light chain and unfolded) NMIIA and NMIIB molecules represent a functional form of NMII in motile cells and that NMIIA and NMIIB copolymerize into nascent bipolar filaments during contractile system assembly. Using subdiffraction stimulated emission depletion microscopy together with a pharmacological block-and-release approach, we report that NMIIA and NMIIB simultaneously incorporate into the cytoskeleton during initiation of contractile system assembly, whereas the characteristic rearward shift of NMIIB relative to NMIIA is established later in the course of NMII turnover.
We show existence of activated NMII monomers in cells, copolymerization of endogenous NMIIA and NMIIB molecules, and contribution of both isoforms, rather than only NMIIA, to early stages of the contractile system assembly. These data change the current paradigms about dynamics and functions of NMII and provide new conceptual insights into the organization and dynamics of the ubiquitous cellular machinery for contraction that acts in multiple cellular contexts.
•Activated unpolymerized NMIIA and NMIIB molecules are directly visualized in cells•NMIIA and NMIIB copolymerize endogenously•NMIIA and NMIIB simultaneously join nascent contractile structures•A posterior shift of NMIIB relative to NMIIA develops during steady-state dynamics
Shutova et al. use platinum replica electron microscopy to show that individual activated (phosphorylated on the regulatory light chain and unfolded) molecules of nonmuscle myosin II (NMII) represent a functional form of NMII in cells and that NMIIA and NMIIB isoforms copolymerize into nascent bipolar filaments during contractile system assembly.
Clamping Mechanism Involved in SNARE-Dependent Exocytosis Giraudo, Claudio G; Eng, William S; Melia, Thomas J ...
Science (American Association for the Advancement of Science),
08/2006, Letnik:
313, Številka:
5787
Journal Article
Recenzirano
During neurotransmitter release at the synapse, influx of calcium ions stimulates the release of neurotransmitter. However, the mechanism by which synaptic vesicle fusion is coupled to calcium has ...been unclear, despite the identification of both the core fusion machinery soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and the principal calcium sensor (synaptotagmin). Here, we describe what may represent a basic principle of the coupling mechanism: a reversible clamping protein (complexin) that can freeze the SNAREpin, an assembled fusion-competent intermediate en route to fusion. When calcium binds to the calcium sensor synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators like synaptotagmin and complexin, can be ectopically expressed on the cell surface. Cells expressing such "flipped" synaptic SNAREs fuse constitutively, but when we coexpressed complexin, fusion was blocked. Adding back calcium triggered fusion from this intermediate in the presence of synaptotagmin.
Membrane fusion between vesicles and target membranes involves the zippering of a four-helix bundle generated by constituent helices derived from target- and vesicle-soluble ...N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In neurons, the protein complexin clamps otherwise spontaneous fusion by SNARE proteins, allowing neurotransmitters and other mediators to be secreted when and where they are needed as this clamp is released. The membrane-proximal accessory helix of complexin is necessary for clamping, but its mechanism of action is unknown. Here, we present experiments using a reconstituted fusion system that suggest a simple model in which the complexin accessory helix forms an alternative four-helix bundle with the target-SNARE near the membrane, preventing the vesicle-SNARE from completing its zippering.
The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, complexin (CPX), ...can act either as a facilitator or as an inhibitor of membrane fusion, constituting a controversial dilemma. Here we take energetic measurements with the surface force apparatus that reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v- and t-SNAREs can engage and then clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9-15 nm but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations of less than 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins.
Upon T-cell activation, the levels of the secondary messenger diacylglycerol (DAG) at the plasma membrane need to be controlled to ensure appropriate T-cell receptor signaling and T-cell functions. ...Extended-Synaptotagmins (E-Syts) are a family of inter-organelle lipid transport proteins that bridge the endoplasmic reticulum and the plasma membrane. In this study, we identify a novel regulatory mechanism of DAG-mediated signaling for T-cell effector functions based on E-Syt proteins. We demonstrate that E-Syts downmodulate T-cell receptor signaling, T-cell-mediated cytotoxicity, degranulation, and cytokine production by reducing plasma membrane levels of DAG. Mechanistically, E-Syt2 predominantly modulates DAG levels at the plasma membrane in resting-state T cells, while E-Syt1 and E-Syt2 negatively control T-cell receptor signaling upon stimulation. These results reveal a previously underappreciated role of E-Syts in regulating DAG dynamics in T-cell signaling.
Synopsis
Extended-Synaptotagmin1 and -2 (E-Syt1 and -2) are lipid transport proteins at ER-plasma membrane contact sites and are responsible for downmodulating DAG levels at the plasma membrane, T cell receptor signaling, and T cell effector functions.
E-Syt proteins regulate T cell receptor signaling and T cell functions through downmodulating DAG levels at the plasma membrane.
E-Syt2 predominantly modulates plasma membrane DAG levels in resting state T cells.
E-Syt1 and E-Syt2 redistribute into DAG-rich regions upon T cell activation.
Extended-Synaptotagmin1 and -2 are lipid transport proteins at ER-plasma membrane contact sites and are responsible for downmodulating DAG levels at the plasma membrane, T cell receptor signaling, and T cell effector functions.
Positron emission tomography is one of the most mature techniques for monitoring the particles range in hadron therapy, aiming to reduce treatment uncertainties and therefore the extent of safety ...margins in the treatment plan. In-beam PET monitoring has been already performed using inter-spill and post-irradiation data, i.e. while the particle beam is off or paused. The full beam acquisition procedure is commonly discarded because the particle spills abruptly increase the random coincidence rates and therefore the image noise. This is because random coincidences cannot be separated by annihilation photons originating from radioactive decays and cannot be corrected with standard random coincidence techniques due to the time correlation of the beam-induced background with the ion beam microstructure. The aim of this paper is to provide a new method to recover in-spill data to improve the images obtained with full-beam PET acquisitions. This is done by estimating the temporal microstructure of the beam and thus selecting input PET events that are less likely to be random ones. The PET detector we used was the one developed within the INSIDE project and tested at the CNAO synchrotron-based facility. The data were taken on a PMMA phantom irradiated with 72 MeV proton pencil beams. The obtained results confirm the possibility of improving the acquired PET data without any external signal coming from the synchrotron or ad hoc detectors.
The crystal structure of complexin bound to a prefusion SNAREpin mimetic shows that the accessory helix extends away from the SNAREpin in an 'open' conformation, binding another SNAREpin and ...inhibiting its assembly, to clamp fusion. In contrast, the accessory helix in the postfusion complex parallels the SNARE complex in a 'closed' conformation. Here we use targeted mutations, FRET spectroscopy and a functional assay that reconstitutes Ca(2+)-triggered exocytosis to show that the conformational switch from open to closed in complexin is needed for synaptotagmin-Ca(2+) to trigger fusion. Triggering fusion requires the zippering of three crucial aspartate residues in the switch region (residues 64-68) of v-SNARE. Conformational switching in complexin is integral to clamp release and is probably triggered when its accessory helix is released from its trans-binding to the neighboring SNAREpin, allowing the v-SNARE to complete zippering and open a fusion pore.
Treatment quality assessment is a crucial feature for both present and next-generation ion therapy facilities. Several approaches are being explored, based on prompt radiation emission or on PET ...signals by Formula: see text-decaying isotopes generated by beam interactions with the body. In-beam PET monitoring at synchrotron-based ion therapy facilities has already been performed, either based on inter-spill data only, to avoid the influence of the prompt radiation, or including both in-spill and inter-spill data. However, the PET images either suffer of poor statistics (inter-spill) or are more influenced by the background induced by prompt radiation (in-spill). Both those problems are expected to worsen for accelerators with improved duty cycle where the inter-spill interval is reduced to shorten the treatment time. With the aim of assessing the detector performance and developing techniques for background reduction, a test of an in-beam PET detector prototype was performed at the CNAO synchrotron-based ion therapy facility in full-beam acquisition modality. Data taken with proton beams impinging on PMMA phantoms showed the system acquisition capability and the resulting activity distribution, separately reconstructed for the in-spill and the inter-spill data. The coincidence time resolution for in-spill and inter-spill data shows a good agreement, with a slight deterioration during the spill. The data selection technique allows the identification and rejection of most of the background originated during the beam delivery. The activity range difference between two different proton beam energies (68 and 72 MeV) was measured and found to be in sub-millimeter agreement with the expected result. However, a slightly longer (2 mm) absolute profile length is obtained for in-spill data when compared to inter-spill data.
Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH), which ...is caused by gene mutations interfering with the cytolytic pathway of cytotoxic T-lymphocytes (CTLs) and natural killer cells. Here we describe a male newborn who met the HLH diagnostic criteria, presented with profound cholestasis, and carried a maternally inherited heterozygous mutation in
, c.568C>T (p.Arg190Cys) in addition to a severe pathogenic variant in
, hemizygous c.1153T>C (Cys385Arg). Although mutations in
gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity outcompeting the effect of endogenous wild-type STXBP2. Interestingly, arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that may act in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied
mutation may have compounded the clinical symptoms; however, the extent by which
deficiency has contributed to HLH in our patient remains unclear.
The aim of this study was to assess the benefits and disadvantages of robot-assisted laparoscopic surgery for disorders of the adrenal gland in terms of feasibility, safety, and length of ...hospitalization.
Twenty consecutive patients with benign lesions of adrenal gland were randomized into two groups: Patients in the laparoscopic group underwent traditional laparoscopic adrenalectomy (LAP), whereas those in the robotic group underwent robot-assisted adrenalectomy (ROBOT) using the da Vinci robotic system.
There was no significant difference between the groups in terms of age, sex, body mass index, and size or locations of lesions. Operative times were significant longer in the ROBOT group (total operative time, 169.2 min range, 136-215 vs 115.3 min (range, 95-155) p < 0.001. Skin-to-skin time was 107 m (range, 77-154) vs 82.1 min (range, 55-120) (p < 0.001). There were no conversions to open surgery. However, conversion to standard laparoscopic surgery was necessary in four of 10 ROBOT patients (40%; left, one right). Perioperative morbidity was higher in the ROBOT group (20% vs 0%). There was no difference in length of hospital stay. In the following ROBOT group, hospital stay was 5.7 days (range, 4-9) vs 5.4 days (range, 4-8) in the LAP group (p = NS). The total cost of the ROBOT procedure ($3,467) was significantly higher than that for LAP ($2,737) (p < 0.01).
Laparoscopic adrenalectomy is superior to robot-assisted adrenalectomy in terms of feasibility, morbidity, and cost.
PDF
