Random, de novo, and conserved proteins: How structure and disorder predictors perform differently.

Understanding the emergence and structural characteristics of de novo and random proteins is crucial for unraveling protein evolution and designing novel enzymes. However, experimental determination of their structures remains challenging. Recent advancements in protein structure prediction, particularly with AlphaFold2 (AF2), have expanded our knowledge of protein structures, but their applicability to de novo and random proteins is unclear. In this study, we investigate the structural predictions and confidence scores of AF2 and protein language model-based predictor ESMFold for de novo and conserved proteins from Drosophila and a dataset of comparable random proteins. We find that the structural predictions for de novo and random proteins differ significantly from conserved proteins. Interestingly, a positive correlation between disorder and confidence scores (pLDDT) is observed for de novo and random proteins, in contrast to the negative correlation observed for conserved proteins. Furthermore, the performance of structure predictors for de novo and random proteins is hampered by the lack of sequence identity. We also observe fluctuating median predicted disorder among different sequence length quartiles for random proteins, suggesting an influence of sequence length on disorder predictions. In conclusion, while structure predictors provide initial insights into the structural composition of de novo and random proteins, their accuracy and applicability to such proteins remain limited. Experimental determination of their structures is necessary for a comprehensive understanding. The positive correlation between disorder and pLDDT could imply a potential for conditional folding and transient binding interactions of de novo and random proteins.

Inhibition of translation termination by the antimicrobial peptide Drosocin.

The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.

Desiccation resistance differences in Drosophila species can be largely explained by variations in cuticular hydrocarbons.

Maintaining water balance is a universal challenge for organisms living in terrestrial environments, especially for insects, which have essential roles in our ecosystem. Although the high surface area to volume ratio in insects makes them vulnerable to water loss, insects have evolved different levels of desiccation resistance to adapt to diverse environments. To withstand desiccation, insects use a lipid layer called cuticular hydrocarbons (CHCs) to reduce water evaporation from the body surface. It has long been hypothesized that the water-proofing capability of this CHC layer, which can confer different levels of desiccation resistance, depends on its chemical composition. However, it is unknown which CHC components are important contributors to desiccation resistance and how these components can determine differences in desiccation resistance. In this study, we used machine-learning algorithms, correlation analyses, and synthetic CHCs to investigate how different CHC components affect desiccation resistance in 50 Drosophila and related species. We showed that desiccation resistance differences across these species can be largely explained by variation in CHC composition. In particular, length variation in a subset of CHCs, the methyl-branched CHCs (mbCHCs), is a key determinant of desiccation resistance. There is also a significant correlation between the evolution of longer mbCHCs and higher desiccation resistance in these species. Given that CHCs are almost ubiquitous in insects, we suggest that evolutionary changes in insect CHC components can be a general mechanism for the evolution of desiccation resistance and adaptation to diverse and changing environments.

Comparative Insecticide Application Techniques (Micro-Sprinkler) Against Drosophila suzukii Matsumura (Diptera: Drosophilidae) in Highbush Blueberry.

Drosophila suzukii (Matsumura), spotted-wing drosophila, is a major pest in small fruit crops including highbush blueberry. Controlling D. suzukii is challenging and chemical control is the main method to manage D. suzukii populations. Growers have expressed interest in using micro-sprinklers as an alternative method to apply insecticides. The current study aimed to evaluate if insecticide applications using micro-sprinklers can be used as an alternative method to protect the fruit from D. suzukii egg-laying. Modeling was used as an additional tool to parameterize the relative insecticide efficacy on oviposition. Field measurements of different treatments were conducted over periods of eleven days on commercial-standard highbush blueberry. Cyantraniliprole and spinetoram were applied using both a micro-sprinkler and a backpack sprayer. Treatments of Chromobacterium subtsugae and zeta-cypermethrin were only applied using a backpack sprayer. Both cyantraniliprole and spinetoram treatments resulted in moderate suppression of D. suzukii egg-laying. No statistical significance was found between micro-sprinkler and backpack sprayer applications for these two insecticides. Zeta-cypermethrin treatments using a backpack sprayer resulted in the most significant suppression of D. suzukii egg-laying over eleven days, while C. subtsugae was less effective at preventing D. suzukii egg-laying. Modeling simulations estimate the impact of the control methods on D. suzukii populations dynamics and simulation outputs indicated that backpack sprayers reduced D. suzukii populations at more pronounced levels compared to micro-sprinkler applications. The present study indicates that there is an underlying value of micro-sprinkler systems as an alternative and rapid spray application technique to help suppress D. suzukii pest populations during high-pressure periods in highbush blueberry production.

Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models.

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.

Drosophila melanogaster Stress Odorant (dSO) Displays the Characteristics of an Interspecific Alarm Cue.

Organisms depend on visual, auditory, and olfactory cues to signal the presence of danger that could impact survival and reproduction. Drosophila melanogaster emits an olfactory alarm signal, termed the Drosophila stress odorant (dSO), in response to mechanical agitation or electric shock. While it has been shown that conspecifics avoid areas previously occupied by stressed individuals, the contextual underpinnings of the emission of, and response to dSO, have received little attention. Using a binary choice assay, we determined that neither age and sex of emitters, nor the time of the day, affected the emission or avoidance of dSO. However, both sex and mating status affected the response to dSO. We also demonstrated that while D. melanogaster, D. simulans, and D. suzukii, have different dSO profiles, its avoidance was not species-specific. Thus, dSO should not be considered a pheromone but a general alarm signal for Drosophila. However, the response levels to both intra- and inter-specific cues differed between Drosophila species and possible reasons for these differences are discussed.

Processing of the capsid proteins of the Betachrysovirus Fusarium graminearum virus-China 9 (FgV-ch9).

While the capsid of viruses in the Alphachrysovirus genus is built of subunits of a single coat protein, the capsid of viruses grouped in the Betachrysovirus genus may consist of subunits of two different proteins. For four of these betachrysoviruses, the detected molecular weights of the putative coat proteins differ from the sizes deduced from the nucleic acid sequence. The origin of these modifications remained unclear and it was hypothesized that the coat proteins undergo unspecific degradation. In our study, we show that these modifications are based on processing steps performed by unknown factors present in extracts of several eukaryotic organisms. Furthermore, we show that the C-terminal domain of P3 is fully degraded after capsid processing and particle assembly.

A new carnivorous plant lineage (Triantha) with a unique sticky-inflorescence trap.

Carnivorous plants consume animals for mineral nutrients that enhance growth and reproduction in nutrient-poor environments. Here, we report that Triantha occidentalis (Tofieldiaceae) represents a previously overlooked carnivorous lineage that captures insects on sticky inflorescences. Field experiments, isotopic data, and mixing models demonstrate significant N transfer from prey to Triantha, with an estimated 64% of leaf N obtained from prey capture in previous years, comparable to levels inferred for the cooccurring round-leaved sundew, a recognized carnivore. N obtained via carnivory is exported from the inflorescence and developing fruits and may ultimately be transferred to next year's leaves. Glandular hairs on flowering stems secrete phosphatase, as seen in all carnivorous plants that directly digest prey. Triantha is unique among carnivorous plants in capturing prey solely with sticky traps adjacent to its flowers, contrary to theory. However, its glandular hairs capture only small insects, unlike the large bees and butterflies that act as pollinators, which may minimize the conflict between carnivory and pollination.

Crystal and solution structures reveal oligomerization of individual capsid homology domains of Drosophila Arc.

Synaptic plasticity is vital for brain function and memory formation. One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids. Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking a large N-terminal domain. Both dArc isoforms are related to the Ty3/gypsy retrotransposon capsid, consisting of N- and C-terminal lobes. Structures of dArc1, as well as capsids formed by both dArc isoforms, have been recently determined. We carried out structural characterization of the four individual dArc lobe domains. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. A truncated N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers, domain-swapped transcription factors, and membrane-interacting BAK domains. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein.

Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins.

Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. Here, we show that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients.

Thermoresponsive motor behavior is mediated by ring neuron circuits in the central complex of Drosophila.

Insects are ectothermal animals that are constrained in their survival and reproduction by external temperature fluctuations which require either active avoidance of or movement towards a given heat source. In Drosophila, different thermoreceptors and neurons have been identified that mediate temperature sensation to maintain the animal's thermal preference. However, less is known how thermosensory information is integrated to gate thermoresponsive motor behavior. Here we use transsynaptic tracing together with calcium imaging, electrophysiology and thermogenetic manipulations in freely moving Drosophila exposed to elevated temperature and identify different functions of ellipsoid body ring neurons, R1-R4, in thermoresponsive motor behavior. Our results show that warming of the external surroundings elicits calcium influx specifically in R2-R4 but not in R1, which evokes threshold-dependent neural activity in the outer layer ring neurons. In contrast to R2, R3 and R4d neurons, thermogenetic inactivation of R4m and R1 neurons expressing the temperature-sensitive mutant allele of dynamin, shibireTS, results in impaired thermoresponsive motor behavior at elevated 31 °C. trans-Tango mediated transsynaptic tracing together with physiological and behavioral analyses indicate that integrated sensory information of warming is registered by neural activity of R4m as input layer of the ellipsoid body ring neuropil and relayed on to R1 output neurons that gate an adaptive motor response. Together these findings imply that segregated activities of central complex ring neurons mediate sensory-motor transformation of external temperature changes and gate thermoresponsive motor behavior in Drosophila.

Analyzing the integrity of oxidative phosphorylation complexes in Drosophila flight muscles.

Drosophila flight muscles are highly enriched with mitochondria and have emerged as a powerful genetic system for studying how oxidative phosphorylation (OXPHOS) complexes are assembled. Here, we describe a series of protocols for analyzing the integrity of OXPHOS complexes in Drosophila via blue native polyacrylamide gel electrophoresis (BN PAGE). We have also included protocols for the additional steps that are typically performed after OXPHOS complexes are separated by BN PAGE, such as Coomassie staining, silver staining, and in-gel OXPHOS activities. For complete details on the use and execution of this protocol, please refer to Murari et al. (2020).

Dynamic structural order of a low-complexity domain facilitates assembly of intermediate filaments.

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent on either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1-I/C IFs functions by forming labile cross-ß interactions. These interactions are seen in polymers made from the tail domain alone, as well as in assembled IFs formed by the intact Tm1-I/C protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology.

Reverse and forward engineering of Drosophila corneal nanocoatings.

Insect eyes have an anti-reflective coating, owing to nanostructures on the corneal surface creating a gradient of refractive index between that of air and that of the lens material1,2. These nanocoatings have also been shown to provide anti-adhesive functionality3. The morphology of corneal nanocoatings are very diverse in arthropods, with nipple-like structures that can be organized into arrays or fused into ridge-like structures4. This diversity can be attributed to a reaction-diffusion mechanism4 and patterning principles developed by Alan Turing5, which have applications in numerous biological settings6. The nanocoatings on insect corneas are one example of such Turing patterns, and the first known example of nanoscale Turing patterns4. Here we demonstrate a clear link between the morphology and function of the nanocoatings on Drosophila corneas. We find that nanocoatings that consist of individual protrusions have better anti-reflective properties, whereas partially merged structures have better anti-adhesion properties. We use biochemical analysis and genetic modification techniques to reverse engineer the protein Retinin and corneal waxes as the building blocks of the nanostructures. In the context of Turing patterns, these building blocks fulfil the roles of activator and inhibitor, respectively. We then establish low-cost production of Retinin, and mix this synthetic protein with waxes to forward engineer various artificial nanocoatings with insect-like morphology and anti-adhesive or anti-reflective function. Our combined reverse- and forward-engineering approach thus provides a way to economically produce functional nanostructured coatings from biodegradable materials.

Functional advantages of Lévy walks emerging near a critical point.

A special class of random walks, so-called Lévy walks, has been observed in a variety of organisms ranging from cells, insects, fishes, and birds to mammals, including humans. Although their prevalence is considered to be a consequence of natural selection for higher search efficiency, some findings suggest that Lévy walks might also be epiphenomena that arise from interactions with the environment. Therefore, why they are common in biological movements remains an open question. Based on some evidence that Lévy walks are spontaneously generated in the brain and the fact that power-law distributions in Lévy walks can emerge at a critical point, we hypothesized that the advantages of Lévy walks might be enhanced by criticality. However, the functional advantages of Lévy walks are poorly understood. Here, we modeled nonlinear systems for the generation of locomotion and showed that Lévy walks emerging near a critical point had optimal dynamic ranges for coding information. This discovery suggested that Lévy walks could change movement trajectories based on the magnitude of environmental stimuli. We then showed that the high flexibility of Lévy walks enabled switching exploitation/exploration based on the nature of external cues. Finally, we analyzed the movement trajectories of freely moving Drosophila larvae and showed empirically that the Lévy walks may emerge near a critical point and have large dynamic range and high flexibility. Our results suggest that the commonly observed Lévy walks emerge near a critical point and could be explained on the basis of these functional advantages.

Alignment and quantification of ChIP-exo crosslinking patterns reveal the spatial organization of protein-DNA complexes.

The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. Applying our approach to collections of ChIP-exo data, we demonstrate that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to analyze Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.

The presence of odd-chain fatty acids in Drosophila phospholipids.

Most fatty acids in phospholipids and other lipid species carry an even number of carbon atoms. Also odd-chain fatty acids (OCFAs), such as C15:0 and C17:0, are widespread throughout the living organism. However, the qualitative and quantitative profiles of OCFAs-containing lipids in living organisms remain unclear. Here, we show that OCFAs are present in Drosophila phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and that their level increases in accordance with progression of growth. Furthermore, we found that food-derived propionic acid/propanoic acid (C3:0) is utilized for production of OCFA-containing PC and PE. This study provides the basis for understanding in vivo function of OCFA-containing phospholipids in development and lipid homeostasis.

A size principle for recruitment of Drosophila leg motor neurons.

To move the body, the brain must precisely coordinate patterns of activity among diverse populations of motor neurons. Here, we use in vivo calcium imaging, electrophysiology, and behavior to understand how genetically-identified motor neurons control flexion of the fruit fly tibia. We find that leg motor neurons exhibit a coordinated gradient of anatomical, physiological, and functional properties. Large, fast motor neurons control high force, ballistic movements while small, slow motor neurons control low force, postural movements. Intermediate neurons fall between these two extremes. This hierarchical organization resembles the size principle, first proposed as a mechanism for establishing recruitment order among vertebrate motor neurons. Recordings in behaving flies confirmed that motor neurons are typically recruited in order from slow to fast. However, we also find that fast, intermediate, and slow motor neurons receive distinct proprioceptive feedback signals, suggesting that the size principle is not the only mechanism that dictates motor neuron recruitment. Overall, this work reveals the functional organization of the fly leg motor system and establishes Drosophila as a tractable system for investigating neural mechanisms of limb motor control.

In the body, spindly nerve cells called motor neurons connect the brain to the muscles. Their role is to control movement, as they translate the electrical signals from the brain into instructions to the muscles. In humans, it takes over 150,000 motor neurons to control the movement of one leg; in contrast, fruit flies only need 50 neurons to operate a leg, despite also executing a variety of movements. Fruit flies are commonly used in laboratories to study an array of biological processes, yet little is known about how their motor neurons direct movements. In particular, it was unclear whether the same principles that control how muscles contract in mammals also applied in the tiny fruit fly. To begin investigating, Azevedo et al. mapped out the arrangement of motor neurons that control muscles in the fruit fly leg. As the leg moved, the activity of both the neurons and the muscles they controlled was recorded, as well as the force that had been generated. The experiments showed that each motor neuron controls a certain range of leg force and speed: some produced small, slow motion important for posture and dexterity, while others created large, fast movements essential to running or escape. In addition, the neurons activate in a particular order ­ cells that control slow movements fire first, and those that direct fast maneuvers later. These processes are also found in other organisms, but the difference is that flies have so few neurons, allowing scientists to reliably identify each motor neuron. Future experiments will therefore be able to test how flies recruit the right neurons to create specific movement sequences. Fruit flies are often used to research human illnesses that affect movement, such as motor neuron disease. A better understanding of the way their neural circuits coordinate the body could help reveal how these conditions emerge.

Ultrasensitive Change in Nucleosome Binding by Multiple Phosphorylations to the Intrinsically Disordered Region of the Histone Chaperone FACT.

Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

Domain Model Explains Propagation Dynamics and Stability of Histone H3K27 and H3K36 Methylation Landscapes.

Chromatin states must be maintained during cell proliferation to uphold cellular identity and genome integrity. Inheritance of histone modifications is central in this process. However, the histone modification landscape is challenged by incorporation of new unmodified histones during each cell cycle, and the principles governing heritability remain unclear. We take a quantitative computational modeling approach to describe propagation of histone H3K27 and H3K36 methylation states. We measure combinatorial H3K27 and H3K36 methylation patterns by quantitative mass spectrometry on subsequent generations of histones. Using model comparison, we reject active global demethylation and invoke the existence of domains defined by distinct methylation endpoints. We find that H3K27me3 on pre-existing histones stimulates the rate of de novo H3K27me3 establishment, supporting a read-write mechanism in timely chromatin restoration. Finally, we provide a detailed quantitative picture of the mutual antagonism between H3K27 and H3K36 methylation and propose that it stabilizes epigenetic states across cell division.