A class VII unconventional myosin is required for phagocytosis

BACKGROUND: Phagocytosis, the process by which cells internalize particles, is essential for the defense of multicellular organisms against invading pathogens and is the major means by which many unicellular organisms obtain nutrients. The actin cytoskeleton plays a critical role in phagocytosis and the observation that a significant amount of force (10-20 nN) is generated during internalization, suggests that a myosin participates in the process. Although more than 15 distinct classes of myosin have been identified, their roles in phagocytosis are unknown. RESULTS: The identification of a class VII unconventional myosin (DdMVII) in the Dictyostelium discoideum amoeba, which is a model for phagocytosis, is reported here. Mutant cells lacking DdMVII exhibited an 80% decrease in the uptake of particles whereas all other actin-based behaviors that were tested, including pinocytosis, exocytosis, cytokinesis and morphogenesis, proceeded normally. The defect in phagocytosis was neither because of altered particle binding nor inability to form actin-filled phagocytic cups. CONCLUSIONS: Molecular genetic analysis of Dictyostelium myosin VII reveals that this motor protein plays a specific and significant role in phagocytosis.     

TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2-Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.     

Characterization of a highly flexible self‐assembling protein system designed to form nanocages

The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(+) and A-(−), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C₃ symmetry. The coiled-coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(+) and A-(−) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(+) and A-(−) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.     

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

An unusual symbiosis, first observed at ∼3000 m depth in the Monterey Submarine Canyon, involves gutless marine polychaetes of the genus Osedax and intracellular endosymbionts belonging to the order Oceanospirillales. Ecologically, these worms and their microbial symbionts have a substantial role in the cycling of carbon from deep-sea whale fall carcasses. Microheterogeneity exists among the Osedax symbionts examined so far, and in the present study the genomes of the two dominant symbionts, Rs1 and Rs2, were sequenced. The genomes revealed heterotrophic versatility in carbon, phosphate and iron uptake, strategies for intracellular survival, evidence for an independent existence, and numerous potential virulence capabilities. The presence of specific permeases and peptidases (of glycine, proline and hydroxyproline), and numerous peptide transporters, suggests the use of degraded proteins, likely originating from collagenous bone matter, by the Osedax symbionts. ¹³C tracer experiments confirmed the assimilation of glycine/proline, as well as monosaccharides, by Osedax. The Rs1 and Rs2 symbionts are genomically distinct in carbon and sulfur metabolism, respiration, and cell wall composition, among others. Differences between Rs1 and Rs2 and phylogenetic analysis of chemotaxis-related genes within individuals of symbiont Rs1 revealed the influence of the relative age of the whale fall environment and support possible local niche adaptation of ‘free-living'' lifestages. Future genomic examinations of other horizontally-propogated intracellular symbionts will likely enhance our understanding of the contribution of intraspecific symbiont diversity to the ecological diversification of the intact association, as well as the maintenance of host diversity.     

Identification of PLX4032‐resistance mechanisms and implications for novel RAF inhibitors

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole‐exome sequencing of drug‐resistant BRAF^V600K melanoma cells. We further describe a new screening approach, a genome‐wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N‐terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF^L505H), is the first resistance‐conferring second‐site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF‐PLX4032 interface with a larger polar residue. Moreover, we show that BRAF^L505H, found in human prostate cancer, is itself a MAPK‐activating, PLX4032‐resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032‐resistant melanoma cells are sensitive to novel, next‐generation BRAF inhibitors, especially the ‘paradox‐blocker’ PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF‐mutations.     

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.     

Tyr-301 Phosphorylation Inhibits Pyruvate Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.     

Computer-aided X-ray screening for tuberculosis and HIV testing among adults with cough in Malawi (the PROSPECT study): A randomised trial and cost-effectiveness analysis

BackgroundSuboptimal tuberculosis (TB) diagnostics and HIV contribute to the high global burden of TB. We investigated costs and yield from systematic HIV-TB screening, including computer-aided digital chest X-ray (DCXR-CAD).Methods and findingsIn this open, three-arm randomised trial, adults (≥18 years) with cough attending acute primary services in Malawi were randomised (1:1:1) to standard of care (SOC); oral HIV testing (HIV screening) and linkage to care; or HIV testing and linkage to care plus DCXR-CAD with sputum Xpert for high CAD4TBv5 scores (HIV-TB screening). Participants and study staff were not blinded to intervention allocation, but investigator blinding was maintained until final analysis. The primary outcome was time to TB treatment. Secondary outcomes included proportion with same-day TB treatment; prevalence of undiagnosed/untreated bacteriologically confirmed TB on day 56; and undiagnosed/untreated HIV. Analysis was done on an intention-to-treat basis. Cost-effectiveness analysis used a health-provider perspective. Between 15 November 2018 and 27 November 2019, 8,236 were screened for eligibility, with 473, 492, and 497 randomly allocated to SOC, HIV, and HIV-TB screening arms; 53 (11%), 52 (9%), and 47 (9%) were lost to follow-up, respectively. At 56 days, TB treatment had been started in 5 (1.1%) SOC, 8 (1.6%) HIV screening, and 15 (3.0%) HIV-TB screening participants. Median (IQR) time to TB treatment was 11 (6.5 to 38), 6 (1 to 22), and 1 (0 to 3) days (hazard ratio for HIV-TB versus SOC: 2.86, 1.04 to 7.87), with same-day treatment of 0/5 (0%) SOC, 1/8 (12.5%) HIV, and 6/15 (40.0%) HIV-TB screening arm TB patients (p = 0.03). At day 56, 2 SOC (0.5%), 4 HIV (1.0%), and 2 HIV-TB (0.5%) participants had undiagnosed microbiologically confirmed TB. HIV screening reduced the proportion with undiagnosed or untreated HIV from 10 (2.7%) in the SOC arm to 2 (0.5%) in the HIV screening arm (risk ratio [RR]: 0.18, 0.04 to 0.83), and 1 (0.2%) in the HIV-TB screening arm (RR: 0.09, 0.01 to 0.71). Incremental costs were US$3.58 and US$19.92 per participant screened for HIV and HIV-TB; the probability of cost-effectiveness at a US$1,200/quality-adjusted life year (QALY) threshold was 83.9% and 0%. Main limitations were the lower than anticipated prevalence of TB and short participant follow-up period; cost and quality of life benefits of this screening approach may accrue over a longer time horizon.ConclusionsDCXR-CAD with universal HIV screening significantly increased the timeliness and completeness of HIV and TB diagnosis. If implemented at scale, this has potential to rapidly and efficiently improve TB and HIV diagnosis and treatment.Trial registrationclinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03519425","term_id":"NCT03519425"}}NCT03519425.

In a randomised trial, Peter MacPherson and colleagues investigate the costs, timeliness, and completeness of computer-aided X-ray screening for tuberculosis and HIV testing in adults with cough in Malawi.