Activation of the receptor KIT induces the secretion of exosome-like small extracellular vesicles.

The receptor tyrosine kinase (RTK) KIT and its ligand stem cell factor (SCF) are essential for human mast cell (huMC) survival and proliferation. HuMCs expressing oncogenic KIT variants secrete large numbers of extracellular vesicles (EVs). The role KIT plays in regulating EV secretion has not been examined.

DELE1 promotes translation-associated homeostasis, growth, and survival in mitochondrial myopathy.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of , and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L).

Endothelial cells release microvesicles that harbour multivesicular bodies and secrete exosomes.

Extracellular vesicles (EVs) released by endothelial cells support vascular homeostasis. To better understand endothelial cell EV biogenesis, we examined cultured human umbilical vein endothelial cells (HUVECs) prepared by rapid freezing, freeze-substitution, and serial thin section electron microscopy (EM). Thin sections of HUVECs revealed clusters of membrane protrusions on the otherwise smooth cell surface.

Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly investigated.

Unique Aggregation of Retroviral Particles Pseudotyped with the Delta Variant SARS-CoV-2 Spike Protein.

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.

The Delta variant SARS-CoV-2 spike protein uniquely promotes aggregation of pseudotyped viral particles.

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay.

CD47 interactions with exportin-1 limit the targeting of mG-modified RNAs to extracellular vesicles.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47 EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs.

CD63 and MHC Class I Subsets of Extracellular Vesicles Produced by Wild-Type and CD47-Deficient Jurkat T Cells Have Divergent Functional Effects on Endothelial Cell Gene Expression.

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay.

The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

The endosomal recycling system dynamically tunes synaptic strength, which underlies synaptic plasticity. Exocytosis is involved in the expression of long-term potentiation (LTP), as postsynaptic cleavage of the SNARE (soluble NSF-attachment protein receptor) protein VAMP2 by tetanus toxin blocks LTP. Moreover, induction of LTP increases the exocytosis of transferrin receptors (TfRs) and markers of recycling endosomes (REs), as well as post-synaptic AMPA type receptors (AMPARs).

A Bimodal Nanosensor for Probing Influenza Fusion Protein Activity Using Magnetic Relaxation.

Viral fusion is a critical step in the entry pathway of enveloped viruses and remains a viable target for antiviral exploration. The current approaches for studying fusion mechanisms include ensemble fusion assays, high-resolution cryo-TEM, and single-molecule fluorescence-based methods. While these methods have provided invaluable insights into the dynamic events underlying fusion processes, they come with their own limitations.

Structural basis for placental malaria mediated by Plasmodium falciparum VAR2CSA.

Plasmodium falciparum VAR2CSA binds to chondroitin sulfate A (CSA) on the surface of the syncytiotrophoblast during placental malaria. This interaction facilitates placental sequestration of malaria parasites resulting in severe health outcomes for both the mother and her offspring. Furthermore, CSA is presented by diverse cancer cells and specific targeting of cells by VAR2CSA may become a viable approach for cancer treatment.

Identifying SARS-CoV-2 Entry Inhibitors through Drug Repurposing Screens of SARS-S and MERS-S Pseudotyped Particles.

While vaccine development will hopefully quell the global pandemic of COVID-19 caused by SARS-CoV-2, small molecule drugs that can effectively control SARS-CoV-2 infection are urgently needed. Here, inhibitors of spike (S) mediated cell entry were identified in a high throughput screen of an approved drugs library with SARS-S and MERS-S pseudotyped particle entry assays. We discovered six compounds (cepharanthine, abemaciclib, osimertinib, trimipramine, colforsin, and ingenol) to be broad spectrum inhibitors for spike-mediated entry.

Macrophage-derived HIV-1 carries bioactive TGF-beta.

Infected T cells and macrophages are the main producers of HIV-1 in infected individuals. Upon release from infected cells, HIV-1 incorporates various cellular membrane proteins, some of which are specific for these cells. However, the functions of cell-encoded proteins in virions remain largely unknown.

Neuronal Activity and Intracellular Calcium Levels Regulate Intracellular Transport of Newly Synthesized AMPAR.

Regulation of AMPA receptor (AMPAR) trafficking is a key modulator of excitatory synaptic transmission; however, intracellular vesicular transport of newly synthesized AMPARs has been little studied due to technical limitations. By combining molecular tools with imaging strategies in cultured rat hippocampal neurons, we found that vesicles containing newly synthesized, GluA1-subunit-containing AMPARs are transported antero- and retrogradely at a mean speed of 1.5 μm.

Zinc and Copper Effects on Stability of Tubulin and Actin Networks in Dendrites and Spines of Hippocampal Neurons.

Zinc and copper ions can modulate the activity of glutamate receptors. However, labile zinc and copper ions likely represent only the tip of the iceberg and other neuronal functions are suspected for these metals in their bound state. We performed synchrotron X-ray fluorescence imaging with 30 nm resolution to image total biometals in dendrites and spines from hippocampal neurons.

Shisa6 traps AMPA receptors at postsynaptic sites and prevents their desensitization during synaptic activity.

Trafficking and biophysical properties of AMPA receptors (AMPARs) in the brain depend on interactions with associated proteins. We identify Shisa6, a single transmembrane protein, as a stable and directly interacting bona fide AMPAR auxiliary subunit. Shisa6 is enriched at hippocampal postsynaptic membranes and co-localizes with AMPARs.

A Septin-Dependent Diffusion Barrier at Dendritic Spine Necks.

Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists.

Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification.

The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons.

Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95.

The spatiotemporal organization of neurotransmitter receptors in postsynaptic membranes is a fundamental determinant of synaptic transmission and information processing by the brain. Using four independent super-resolution light imaging methods and EM of genetically tagged and endogenous receptors, we show that, in rat hippocampal neurons, AMPARs are often highly concentrated inside synapses into a few clusters of ∼70 nm that contain ∼20 receptors. AMPARs are stabilized reversibly in these nanodomains and diffuse freely outside them.

Mass of the postsynaptic density and enumeration of three key molecules.

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.

Spike structure at the interface between gliding Mycoplasma mobile cells and glass surfaces visualized by rapid-freeze-and-fracture electron microscopy.

Mycoplasma mobile is a flask-shaped bacteria that binds to a substrate and glides towards its tapered end, the so-called "head-like protrusion," by an unknown mechanism. To search for cellular structures underlying this motility, the cell-substrate interface of actively gliding cells was visualized by rapid-freeze-and-freeze-fracture rotary-shadow electron microscopy. Novel structures, called "spikes," were observed to protrude from the cell membrane and attach to the glass surface at their distal end.

Distribution of postsynaptic density (PSD)-95 and Ca2+/calmodulin-dependent protein kinase II at the PSD.

Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We determined the positions of calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95 within the three-dimensional structure of isolated PSDs using immunogold labeling, rotary shadowing, and electron microscopic tomography. The results show that all PSDs contain a central mesh immediately underlying the postsynaptic membrane.