An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission.

The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity.

CHMP4B contributes to maintaining the follicular cells integrity in the panoistic ovary of the cockroach Blattella germanica.

BACKGROUND: The Endosomal Sorting Complex Required for Transport (ESCRT) is a highly conserved cellular machinery essential for many cellular functions, including transmembrane protein sorting, endosomal trafficking, and membrane scission. CHMP4B is a key component of ESCRT-III subcomplex and has been thoroughly studied in the meroistic ovaries of Drosophila melanogaster showing its relevance in maintaining this reproductive organ during the life of the fly. However, the role of the CHMP4B in the most basal panoistic ovaries remains elusive. RESULTS: Using RNAi, we examined the function of CHMP4B in the ovary of Blattella germanica in two different physiological stages: in last instar nymphs, with proliferative follicular cells, and in vitellogenic adults when follicular cells enter in polyploidy and endoreplication. In Chmp4b-depleted specimens, the actin fibers change their distribution, appearing accumulated in the basal pole of the follicular cells, resulting in an excess of actin bundles that surround the basal ovarian follicle and modifying their shape. Depletion of Chmp4b also determines an actin accumulation in follicular cell membranes, resulting in different cell morphologies and sizes. In the end, these changes disrupt the opening of intercellular spaces between the follicular cells (patency) impeding the incorporation of yolk proteins to the growing oocyte and resulting in female sterility. In addition, the nuclei of follicular cells appeared unusually elongated, suggesting an incomplete karyokinesis. CONCLUSIONS: These results proved CHMP4B essential in preserving the proper expression of cytoskeleton proteins vital for basal ovarian follicle growth and maturation and for yolk protein incorporation. Moreover, the correct distribution of actin fibers in the basal ovarian follicle emerged as a critical factor for the successful completion of ovulation and oviposition. SIGNIFICANCE: The overall results, obtained in two different proliferative stages, suggest that the requirement of CHMP4B in B. germanica follicular epithelium is not related to the proliferative stage of the tissue.

The ESCRT-III complex contributes to macromitophagy in yeast.

Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, glutathione-S-transferase (GST) pulldown and bimolecular fluorescence complementation (BiFC) assay, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32-marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.

RNA interference-mediated control of cigarette beetle, Lasioderma serricorne.

The cigarette beetle (CB; Lasioderma serricorne) is a pest on many stored products including tobacco. Fumigation is the common control method currently used. However, the options for controlling this pest are limited, due to resistance issues and phasing out of currently used chemical insecticides. Here, we evaluated RNA interference (RNAi) as a potential method for controlling the CB. RNA isolated from different stages was sequenced and assembled into a transcriptome. The CB RNA sequences showed the highest homology with those in the red flour beetle, Tribolium castaneum. Orthologs of proteins known to function in RNAi pathway were identified in the CB transcriptome, suggesting that RNAi may work well in this insect. Also, 32 P-labeled double-stranded RNA (dsRNA) injected into CB larvae and adults was processed to small interference RNAs. We selected 12 genes that were shown to be the effective RNAi targets in T. castaneum and other insects and identified orthologs of them in the CB by searching its transcriptome. Injection of dsRNA targeting genes coding for GAWKY, Kinesin, Sec23, SNF7, and 26S proteasome subunit 6B into the CB larvae caused 100% mortality. Feeding dsRNA targeting SNF7 and 26S proteasome subunit 6B by sucrose droplet assay induced more than 90% mortality, which is 1.8 times higher than the mortality induced by dsGFP control (53%). These data demonstrate an efficient RNAi response in CB, suggesting that RNAi could be developed as an efficient method to control this pest.

Crucial role of Snf7-3 in synaptic function and cognitive behavior revealed by conventional and conditional knockout mouse models.

Snf7-3 is a crucial component of the endosomal sorting complexes required for transport (ESCRT) pathway, playing a vital role in endolysosomal functions. To elucidate the role of Snf7-3 in vivo, we developed conventional-like and conditional Snf7-3 knockout (KO) mouse models using a "Knockout-first" strategy. Conventional-like Snf7-3 KO mice showed significantly reduced Snf7-3 mRNA expression, and older mice (25-40 weeks) exhibited impaired social recognition and increased miniature excitatory postsynaptic currents (mEPSCs). Similarly, conditional KO mice aged 8-24 weeks, with Snf7-3 specifically deleted in forebrain excitatory neurons, displayed impaired object location memory and elevated mEPSC frequency. Consistently, Snf7-3 knockdown in cultured mouse hippocampal neurons led to increased densities of pre- and postsynaptic puncta, supporting the observed increase in mEPSC frequency. In addition, enhanced dendritic complexity was observed in the medial prefrontal cortex of these mice, indicating early synaptic disturbances. Our findings underscore the critical role of Snf7-3 in maintaining normal cognitive functions and social behaviors. The observed synaptic and behavioral deficits in both conventional-like and conditional KO mice highlight the importance of Snf7-3 in specific neuronal populations, suggesting that early synaptic changes could precede more pronounced cognitive impairments.

Structural Analysis of the ESCRT-III Regulator Lethal(2) Giant Discs/Coiled-Coil and C2 Domain-Containing Protein 1 (Lgd/CC2D1).

Members of the LGD/CC2D1 protein family contain repeats of the family-defining DM14 domains. Via this domain, they interact with members of the CHMP family, which are essential for the ESCRT machinery-mediated formation of intraluminal vesicles during endosome maturation. Here, we investigate the requirement of the DM14 domains for the function of Lgd in detail. We found that although both odd-numbered DM14s can act in a functionally redundant manner, the redundancy is not complete and both contribute to the full function of Lgd. Our analysis indicates that some of the AAs that form the KARRxxR motif of the onDM14s are not exchangeable by similarly charged AAs without loss of function, indicating that they not only provide charge, but also fulfil structural roles. Furthermore, we show that the region of Lgd between DM14-4 and the C2 domain as well as its C-terminal region to the C2 domain are important for protein stability/function. Moreover, we analysed the importance of AAs that are conserved in all DM14 domains. Finally, our analysis of the C. elegans ortholog of Lgd revealed that it has only one DM14 domain that is functionally equivalent to the onDM14s. Altogether, the results further the understanding of how Lgd family members regulate the ESCRT machinery.

Vtc4 Promotes the Entry of Phagophores into Vacuoles in the Saccharomyces cerevisiae Snf7 Mutant Cell.

Endocytosis and autophagy are the main pathways to deliver cargoes in vesicles and autophagosomes, respectively, to vacuoles/lysosomes in eukaryotes. Multiple positive regulators but few negative ones are reported to regulate the entry of vesicles and autophagosomes into vacuoles/lysosomes. In yeast, the Rab5 GTPase Vps21 and the ESCRT (endosomal sorting complex required for transport) are positive regulators in endocytosis and autophagy. During autophagy, Vps21 regulates the ESCRT to phagophores (unclosed autophagosomes) to close them. Phagophores accumulate on vacuolar membranes in both vps21∆ and ESCRT mutant cells under a short duration of nitrogen starvation. The vacuolar transport chaperon (VTC) complex proteins are recently found to be negative regulators in endocytosis and autophagy. Phagophores in vps21∆ cells are promoted to enter vacuoles when the VTC complex proteins are absent. Phagophores are easily observed inside vacuoles when any of these VTC complex proteins (Vtc1, 2, 4, 5) are removed. However, it is unknown whether the removal of VTC complex proteins will also promote the entry of phagophores into vacuoles in ESCRT mutant cells under the same conditions. Snf7 is a core subunit of ESCRT subcomplex III (ESCRT-III), and phagophores accumulate in snf7∆ cells under a short duration of nitrogen starvation. We used green fluorescence protein (GFP) labeled Atg8 to display phagophores and FM4-64-stained or Vph1-GFP-labeled membrane structures to show vacuoles, then examined fluorescence localization and GFP-Atg8 degradation in snf7∆ and snf7∆vtc4∆ cells. Results showed that Vtc4 depletion promoted the entry of phagophores in snf7∆ cells into vacuoles as it did for vps21∆ cells, although the promotion level was more obvious in vps21∆ cells. This observation indicates that the VTC complex proteins may have a widespread role in negatively regulating cargos to enter vacuoles in yeast.

Using GBP Nanotrap to Restore Autophagy in the Rab5/Vps21 Mutant by Forcing Snf7 and Atg17 Interaction.

Protein-protein interactions are important for physiology performance. Green fluorescent protein (GFP) is a widely used protein tag to show protein localization in vivo. GFP binding protein (GBP) is a specific domain with high affinity to GFP. A novel technique with GBP fused protein X tagged with red fluorescence protein binding to GFP of GFP fused protein Y to establish a close association for proteins X and Y independently from other proteins has recently been developed. It is found that the interaction and colocalization between Snf7 and Atg17 is impaired in Saccharomyces cerevisiae vps21Δ cells, which are defective in autophagy. In order to determine whether the interaction between Snf7 and Atg17 is important for autophagy, we forced the interaction between Snf7 and Atg17 through GBP-GFP binding. Snf7-GBP-mCherry and/or GFP-Atg17 tagged wild-type and vps21Δ cells were compared for autophagy process under starvation by determining the maturation of proprotein of Ape1 (prApe1). Our results showed that the defective autophagy in vps21Δ cells was significantly suppressed when both Snf7-GBP-mCherry and GFP-Atg17 were installed. Our results indicate that the GBP-GFP nanotrap technique is a powerful tool to restore colocalization/interaction in vivo and the Snf7-Atg17 interaction is important for yeast autophagy.

Mechanisms of negative membrane curvature sensing and generation by ESCRT III subunit Snf7.

Certain proteins have the propensity to bind to negatively curved membranes and generate negative membrane curvature. The mechanism of action of these proteins is much less studied and understood than those that sense and generate positive curvature. In this work, we use implicit membrane modeling to explore the mechanism of an important negative curvature sensing and generating protein: the main ESCRT III subunit Snf7. We find that Snf7 monomers alone can sense negative curvature and that curvature sensitivity increases for dimers and trimers. We have observed spontaneous bending of Snf7 oligomers into circular structures with preferred radius of ~20 nm. The preferred curvature of Snf7 filaments is further confirmed by the simulations of preformed spirals on a cylindrical membrane surface. Snf7 filaments cannot bind with the same interface to flat and curved membranes. We find that even when a filament has the preferred radius, it is always less stable on the flat membrane surface than on the interior cylindrical membrane surface. This provides an additional energy for membrane bending which has not been considered in the spiral spring model. Furthermore, the rings on the cylindrical spirals are bridged together by helix 4 and hence are extra stabilized compared to the spirals on the flat membrane surface.

ESCRTing in cereals: still a long way to go.

The multivesicular body (MVB) sorting pathway provides a mechanism for the delivery of cargo destined for degradation to the vacuole or lysosome. The endosomal sorting complex required for transport (ESCRT) is essential for the MVB sorting pathway by driving the cargo sorting to its destination. Many efforts in plant research have identified the ESCRT machinery and functionally characterised the first plant ESCRT proteins. However, most studies have been performed in the model plant Arabidopsis thaliana that is genetically and physiologically different to crops. Cereal crops are important for animal feed and human nutrition and have further been utilized as promising candidates for recombinant protein production. In this review, I summarize the role of plant ESCRT components in cereals that are involved in efficient adaptation to environmental stress and grain development. A special focus is on barley (Hordeum vulgare L.) ESCRT proteins, where recent studies show their quantitative mapping during grain development, e.g. associating HvSNF7.1 with protein trafficking to protein bodies (PBs) in starchy endosperm. Thus, it is indispensable to identify the molecular key-players within the endomembrane system including ESCRT proteins to optimize and possibly enhance tolerance to environmental stress, grain yield and recombinant protein production in cereal grains.

Genetic and Biochemical Analyses of Yeast ESCRT.

Budding yeast Saccharomyces cerevisiae is an ideal model organism to study membrane trafficking pathways. The ESCRT (endosomal sorting complexes required for transport) pathway was first identified in this organism. Upon recognition of endocytosed ubiquitinated membrane proteins at endosomes, ESCRTs assemble at these organelles to catalyze the biogenesis of multivesicular bodies (MVBs). Formation of MVBs leads to the trafficking of these membrane proteins to vacuoles for degradation. Here, we describe genetic and biochemical approaches to study ESCRT function. We outline in vivo endocytosis assays using two model cargoes in Saccharomyces cerevisiae and also describe an in vitro approach to analyze ESCRT-III polymerization on lipid monolayers.

Electrostatic lateral interactions drive ESCRT-III heteropolymer assembly.

Self-assembly of ESCRT-III complex is a critical step in all ESCRT-dependent events. ESCRT-III hetero-polymers adopt variable architectures, but the mechanisms of inter-subunit recognition in these hetero-polymers to create flexible architectures remain unclear. We demonstrate in vivo and in vitro that the Saccharomyces cerevisiae ESCRT-III subunit Snf7 uses a conserved acidic helix to recruit its partner Vps24. Charge-inversion mutations in this helix inhibit Snf7-Vps24 lateral interactions in the polymer, while rebalancing the charges rescues the functional defects. These data suggest that Snf7-Vps24 assembly occurs through electrostatic interactions on one surface, rather than through residue-to-residue specificity. We propose a model in which these cooperative electrostatic interactions in the polymer propagate to allow for specific inter-subunit recognition, while sliding of laterally interacting polymers enable changes in architecture at distinct stages of vesicle biogenesis. Our data suggest a mechanism by which interaction specificity and polymer flexibility can be coupled in membrane-remodeling heteropolymeric assemblies.

Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens.

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes.

Sublethal Effects of vATPase-A and Snf7 dsRNAs on Biology of Southern Corn Rootworm, Diabrotica undecimpunctata howardi Barber.

RNA interference is a powerful tool against corn rootworm. Adults and neonates of southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), were exposed to the LC50 of vATPase-A and Snf7 double-stranded RNAs (dsRNAs), and the effects on female fecundity, egg viability, male fitness as measured by sperm viability and mating capacity, larval recovery along with dry weight, and instar determination 10 d after exposure to dsRNA, were determined. Significant reductions were observed for a number of parameters in dsRNA-exposed rootworms relative to control treatments. Female fecundity and larval recovery were significantly reduced after exposure to both dsRNAs. In addition, larval dry weight and recovery of 2nd and 3rd instars along with dry weight for 3rd instars were significantly reduced after neonate exposure to vATPase-A dsRNA. Neither dsRNA affected male capacity to mate or sperm viability after exposure to the respective LC50s. After 10 d of feeding on untreated corn roots, neonates that survived exposure for 2 d to the vATPase-A dsRNA LC50 exhibited lower dry weight than the control. There was significant gene knockdown in adult males and females after exposure for 5 d to LC50 of vATPase-A and Snf7 dsRNAs. The parameters are discussed in terms of fitness and possible outcomes after deployment of corn hybrids expressing dsRNAs.

Structural Basis for Regulation of ESCRT-III Complexes by Lgd.

The ESCRT-III complex induces outward membrane budding and fission through homotypic polymerization of its core component Shrub/CHMP4B. Shrub activity is regulated by its direct interaction with a protein called Lgd in flies, or CC2D1A or B in humans. Here, we report the structural basis for this interaction and propose a mechanism for regulation of polymer assembly. The isolated third DM14 repeat of Lgd binds Shrub, and an Lgd fragment containing only this DM14 repeat and its C-terminal C2 domain is sufficient for in vivo function. The DM14 domain forms a helical hairpin with a conserved, positively charged tip, that, in the structure of a DM14 domain-Shrub complex, occupies a negatively charged surface of Shrub that is otherwise used for homopolymerization. Lgd mutations at this interface disrupt its function in flies, confirming functional importance. Together, these data argue that Lgd regulates ESCRT activity by controlling access to the Shrub self-assembly surface.

ESCRT-III activation by parallel action of ESCRT-I/II and ESCRT-0/Bro1 during MVB biogenesis.

The endosomal sorting complexes required for transport (ESCRT) pathway facilitates multiple fundamental membrane remodeling events. Previously, we determined X-ray crystal structures of ESCRT-III subunit Snf7, the yeast CHMP4 ortholog, in its active and polymeric state (Tang et al., 2015). However, how ESCRT-III activation is coordinated by the upstream ESCRT components at endosomes remains unclear. Here, we provide a molecular explanation for the functional divergence of structurally similar ESCRT-III subunits. We characterize novel mutations in ESCRT-III Snf7 that trigger activation, and identify a novel role of Bro1, the yeast ALIX ortholog, in Snf7 assembly. We show that upstream ESCRTs regulate Snf7 activation at both its N-terminal core domain and the C-terminus α6 helix through two parallel ubiquitin-dependent pathways: the ESCRT-I-ESCRT-II-Vps20 pathway and the ESCRT-0-Bro1 pathway. We therefore provide an enhanced understanding for the activation of the spatially unique ESCRT-III-mediated membrane remodeling.

Chitosan, Carbon Quantum Dot, and Silica Nanoparticle Mediated dsRNA Delivery for Gene Silencing in Aedes aegypti: A Comparative Analysis.

In spite of devastating impact of mosquito borne pathogens on humans, widespread resistance to chemical insecticides and environmental concerns from residual toxicity limit mosquito control strategies. We tested three nanoparticles, chitosan, carbon quantum dot (CQD), and silica complexed with dsRNA, to target two mosquito genes (SNF7 and SRC) for controlling Aedes aegypti larvae. Relative mRNA levels were quantified using qRT-PCR to evaluate knockdown efficiency in nanoparticle-dsRNA treated larvae. The knockdown efficiency of target genes correlated with dsRNA mediated larval mortality. Among the three nanoparticles tested, CQD was the most efficient carrier for dsRNA retention, delivery, and thereby causing gene silencing and mortality in Ae. aegypti.

Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments.

The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30 Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling.