Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.

Dynamic actin/septin network in megakaryocytes coordinates proplatelet elaboration.

Megakaryocytes (MK) undergo extensive cytoskeletal rearrangements as they give rise to platelets. While cortical microtubule sliding has been implicated in proplatelet formation, the role of the actin cytoskeleton in proplatelet elongation is less understood. It is assumed that actin filament reorganization is important for platelet generation given that mouse models with mutations in actin-associated proteins exhibit thrombocytopenia. However, due to the essential role of the actin network during MK development, a differential understanding of the contribution of the actin cytoskeleton on proplatelet release is lacking. Here, we reveal that inhibition of actin polymerization impairs the formation of elaborate proplatelets by hampering proplatelet extension and bead formation along the proplatelet shaft, which was mostly independent of changes in cortical microtubule sliding. We identify Cdc42 and its downstream effectors, septins, as critical regulators of intracellular actin dynamics in MK, inhibition of which, similarly to inhibition of actin polymerization, impairs proplatelet movement and beading. Super-resolution microscopy revealed a differential association of distinctive septins with the actin and microtubule cytoskeleton, respectively, which was disrupted upon septin inhibition and diminished intracellular filamentous actin dynamics. In vivo, septins, similarly to F-actin, were subject to changes in expression upon enforcing proplatelet formation through prior platelet depletion. In summary, we demonstrate that a Cdc42/septin axis is not only important for MK maturation and polarization, but is further required for intracellular actin dynamics during proplatelet formation.

Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion.

As with most intermediate filament systems, the hierarchical self-assembly of vimentin into nonpolar filaments requires no nucleators or energy input. Utilizing a set of live-cell, single-molecule, and super-resolution microscopy tools, here we show that in mammalian cells, the assembly and disassembly of the vimentin cytoskeleton is highly sensitive to the protein net charge state. Starting with the intriguing observation that the vimentin cytoskeleton fully disassembles under hypotonic stress yet reassembles within seconds upon osmotic pressure recovery, we pinpoint ionic strength as its underlying driving factor. Further modulating the pH and expressing differently charged constructs, we converge on a model in which the vimentin cytoskeleton is destabilized by Coulomb repulsion when its mass-accumulated negative charges (-18 per vimentin protein) along the filament are less screened or otherwise intensified, and stabilized when the charges are better screened or otherwise reduced. Generalizing this model to other intermediate filaments, we further show that whereas the negatively charged GFAP cytoskeleton is similarly subject to fast disassembly under hypotonic stress, the cytokeratin, as a copolymer of negatively and positively charged subunits, does not exhibit this behavior. Thus, in cells containing both vimentin and keratin cytoskeletons, hypotonic stress disassembles the former but not the latter. Together, our results both provide new handles for modulating cell behavior and call for new attention to the effects of net charges in intracellular protein interactions.

Ectopic Expression of a Truncated Isoform of Hair Keratin 81 in Breast Cancer Alters Biophysical Characteristics to Promote Metastatic Propensity.

Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.

Controlling the Nature of Etched Si Nanostructures: High- versus Low-Load Metal-Assisted Catalytic Etching (MACE) of Si Powders.

Metal-assisted catalytic etching (MACE) involving Ag deposited on Si particles has been reported as a facile method for the production of Si nanowires (Si NWs). We show that the structure of Si particles subjected to MACE changes dramatically in response to changing the loading of the Ag catalyst. The use of acetic acid as a surfactant and controlled injection of AgNO3(aq) enhanced Ag deposition. The use of acetic acid and controlled injection of H2O2 not only facilitated optimization of the etching step but also allowed us to identify a previously unobserved etching regime that we denote as low-load MACE (LL-MACE). Material produced by LL-MACE exhibits dramatically different yield and structural characteristics as compared to conventionally produced material. We demonstrate the production of Si NWs as well as mesoporous Si nanoparticles from an inexpensive metallurgical-grade Si powder. High loading of Ag (HL-MACE) generates parallel etch track pores created by the correlated motion of Ag nanoparticles. The uniform size distribution (predominantly 70-100 nm) of the Ag nanoparticles is generated dynamically during etching. The walls of these etch track pores are cleaved readily by ultrasonic agitation to form Si NWs. Low loading of Ag (LL-MACE) creates 10-50 nm Ag nanoparticles that etch in an uncorrelated (randomly directed) fashion to generate a bimodal distribution of mesoporosity peaking at ∼4 and 13-21 nm. The use of a syringe pump to deliver the oxidant (H2O2) and Ag+ is essential for increased product uniformity and yield. Different process temperatures and grades of Si produced significantly different pore size distributions. These results facilitate the production of Si NWs and mesoporous nanoparticles with high yield, low cost, and controlled properties that are suitable for applications in, e.g., lithium-ion batteries, drug delivery, as well as biomedical imaging and contrast enhancement.

Low-Load Metal-Assisted Catalytic Etching Produces Scalable Porosity in Si Powders.

The recently discovered low-load metal-assisted catalytic etching (LL-MACE) creates nanostructured Si with controllable and variable characteristics that distinguish this technique from the conventional high-load variant. LL-MACE employs 150 times less metal catalyst and produces porous Si instead of Si nanowires. In this work, we demonstrate that some of the features of LL-MACE cannot be explained by the present understanding of MACE. With mechanistic insight derived from extensive experimentation, it is demonstrated that (1) the method allows the use of not only Ag, Pd, Pt, and Au as metal catalysts but also Cu and (2) judicious combinations of process parameters such as the type of metal, Si doping levels, and etching temperatures facilitate control over yield (0.065-88%), pore size (3-100 nm), specific surface area (20-310 m2·g-1), and specific pore volume (0.05-1.05 cm3·g-1). The porous structure of the product depends on the space-charge layer, which is controlled by the Si doping and the chemical identity of the deposited metal. The porous structure was also dependent on the dynamic structure of the deposited metal. A distinctive comet-like structure of metal nanoparticles was observed after etching with Cu, Ag, Pd, and, in some cases, Pt; this structure consisted of 10-50 nm main particles surrounded by smaller (<5 nm) nanoparticles. With good scalability and precise control of structural properties, LL-MACE facilitates Si applications in photovoltaics, energy storage, biomedicine, and water purification.

Crystallographically Determined Etching and Its Relevance to the Metal-Assisted Catalytic Etching (MACE) of Silicon Powders.

Metal-assisted catalytic etching (MACE) using Ag nanoparticles as catalysts and H2O2 as oxidant has been performed on single-crystal Si wafers, single-crystal electronics grade Si powders, and polycrystalline metallurgical grade Si powders. The temperature dependence of the etch kinetics has been measured over the range 5-37°C. Etching is found to proceed preferentially in a 〈001〉 direction with an activation energy of ~0.4 eV on substrates with (001), (110), and (111) orientations. A quantitative model to explain the preference for etching in the 〈001〉 direction is developed and found to be consistent with the measured activation energies. Etching of metallurgical grade powders produces particles, the surfaces of which are covered primarily with porous silicon (por-Si) in the form of interconnected ridges. Silicon nanowires (SiNW) and bundles of SiNW can be harvested from these porous particles by ultrasonic agitation. Analysis of the forces acting between the metal nanoparticle catalyst and the Si particle demonstrates that strongly attractive electrostatic and van der Waals interactions ensure that the metal nanoparticles remain in intimate contact with the Si particles throughout the etch process. These attractive forces draw the catalyst toward the interior of the particle and explain why the powder particles are etched equivalently on all the exposed faces.