Budding Events Research Articles

A computer vision and residual neural network (ResNet) combined method for automated and accurate yeast replicative aging analysis of high-throughput microfluidic single-cell images

With the rapid development of microfluidic platforms in high-throughput single-cell culturing, laborious operation to manipulate massive budding yeast cells (Saccharomyces cerevisiae) in replicative aging studies has been greatly simplified and automated. As a result, large datasets of microscopy images bring challenges to fast and accurately determine yeast replicative lifespan (RLS), which is the most important parameter to study cell aging. Based on our microfluidic diploid yeast long-term culturing (DYLC) chip that features 1100 traps to immobilize single cells and record their proliferation and aging via time-lapse imaging, herein, a dedicated algorithm combined with computer vision and residual neural network (ResNet) was presented to efficiently process tremendous micrographs in a high-throughput and automated manner. The image-processing algorithm includes following pivotal steps: (i) segmenting multi-trap micrographs into time-lapse single-trap sub-images, (ii) labeling 8 yeast budding features and training the 18-layer ResNet, (iii) converting the ResNet predictions in analog values into digital signals, (iv) recognizing cell dynamic events, and (v) determining yeast RLS and budding time interval (BTI) ultimately. The ResNet algorithm achieved high F1 scores (over 92%) demonstrating the effectiveness and accuracy in the recognition of yeast budding events, such as bud appearance, daughter dissection and cell death. Therefore, the results conduct that similar deep learning algorithms could be tailored to analyze high-throughput microscopy images and extract multiple cell behaviors in microfluidic single-cell analysis.

Fluorescence Resonance Energy Transfer to Detect Plasma Membrane Perturbations in Giant Plasma Membrane Vesicles.

Disruptions and perturbations of the cellular plasma membrane by peptides have garnered significant interest in the elucidation of biological phenomena. Typically, these complex processes are studied using liposomes as model membranes-either by encapsulating a fluorescent dye or by other spectroscopic approaches, such as nuclear magnetic resonance. Despite incorporating physiologically relevant lipids, no synthetic model truly recapitulates the full complexity and molecular diversity of the plasma membrane. Here, biologically representative membrane models, giant plasma membrane vesicles (GPMVs), are prepared from eukaryotic cells by inducing a budding event with a chemical stressor. The GPMVs are then isolated, and bilayers are labelled with fluorescent lipophilic tracers and incubated in a microplate with a membrane-active peptide. As the membranes become damaged and/or aggregate, the resulting fluorescence resonance energy transfer (FRET) between the two tracers increases and is measured periodically in a microplate. This approach offers a particularly useful way to detect perturbations when the membrane complexity is an important variable to consider. Additionally, it provides a way to kinetically detect damage to the plasma membrane, which can be correlated with the kinetics of peptide self-assembly or structural rearrangements. Key features • Allows testing of various peptide-membrane interaction conditions (peptide:phospholipid ratio, ionic strength, buffer, etc.) at once. • Uses intact plasma membrane vesicles that can be prepared from a variety of cell lines. • Can offer comparable throughput as with traditional synthetic lipid models (e.g., dye-encapsulated liposomes).

Kleptoparasites of social spider colonies do not track hosts' subdivided population structure.

Organisms with lower dispersal abilities tend to have more genetically dissimilar populations. The same is true for parasites, whose transmission frequency may depend on the population structure of the host. This should be especially true when hosts and parasites face similar barriers to dispersal. Here, we considered the similarities between host and parasite population structure in a social spider system. In this system, host colonies are typified by rapid growth via internal recruitment followed by budding or fission events when colonies grow too large, with each colony representing a distinct population. Host colonies provide habitat for kleptoparasitic spiders, which steal prey from and may also feed directly on host individuals. We asked whether kleptoparasites exhibit a similar degree of population subdivision as their host. Under the free-mixing hypothesis (i.e., horizontal transmission), kleptoparasites would have well-mixed populations across broader regions than a single host nest, whereas host populations would be strongly genetically structured. Under the host-tracking hypothesis (i.e., vertical transmission), kleptoparasites would have a population structure that parallels that of the host. We conducted a genotype-by-sequencing study to assess the population structure of both hosts and kleptoparasites within three nearby regions in eastern Ecuador. We found strong signatures of population differentiation and bottlenecks in the host species, which is congruent with past studies. However, we found that kleptoparasite populations were well mixed across host nests, with no evidence of recent bottlenecks. These results support our free-mixing hypothesis, suggesting that kleptoparasites follow patterns of horizontal transmission in this social spider system.

Analysis of individual HIV-1 budding event using fast AFM reveals a multiplexed role for VPS4

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Although the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (∼100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wild-type (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 38% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

Stochastic diffusion characterises early colony formation in Mediterranean coral Corallium rubrum

The colony formation in Mediterranean coral Corallium rubrum is initiated by a larva that metamorphoses into the first polyp of the emerging colony approximately two weeks after settlement. The primary polyp then sets up a slow process that eventually, at least after a few years, gives rise to a tree-like rigid colony structure on which other polyps flourish. For a mature colony, this axial skeleton provides support for new polyps. However, the first emergence of the characteristic axial skeleton can take two years or more from the larva stage. The early colony morphology, instead, is shaped exclusively by the polyps’ abundant deposition of sclerites, a magnesian calcite biomineral that has a different granularity from the distinctive red-coloured skeleton. With the appearance of the first polyp, a growing sclerite heap in a mesoglea layer provides a base for the emerging colony. In this paper, to elucidate the mechanical processes of early skeleton development in C. rubrum colonies, we present a computational model whereby the mesoglea layer provides a diffusion medium for the sclerites that the polyps deposit. We show that our stochastic model with three parameters captures the dynamic variability observed in measurements on living colonies. Our simulation results provide evidence for a diffusion process whereby the interplay between polyp budding and sclerite deposition are the main determinants of structure in early colony formation. Our model demonstrates that the frequency of budding events in an early colony can be described as a function of the available mesoglea surface whereas the number of polyps on the colony plays a secondary role in determining this frequency. We show that these model predictions are confirmed by direct observations on the colonies in our sample. Moreover, our results indicate that diffusion is a prevalent mechanism of colony development also at later stages of a colony’s life span.

Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis.

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

YeastMate: neural network-assisted segmentation of mating and budding events in Saccharomyces cerevisiae.

SummaryHere, we introduce YeastMate, a user-friendly deep learning-based application for automated detection and segmentation of Saccharomyces cerevisiae cells and their mating and budding events in microscopy images. We build upon Mask R-CNN with a custom segmentation head for the subclassification of mother and daughter cells during lifecycle transitions. YeastMate can be used directly as a Python library or through a standalone application with a graphical user interface (GUI) and a Fiji plugin as easy-to-use frontends. Availability and implementationThe source code for YeastMate is freely available at https://github.com/hoerlteam/YeastMate under the MIT license. We offer installers for our software stack for Windows, macOS and Linux. A detailed user guide is available at https://yeastmate.readthedocs.io.Supplementary information Supplementary data are available at Bioinformatics online.

Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses.

Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.

Developmental Process of a Heterozooid: Avicularium Formation in a Bryozoan, Bugulina californica.

In bryozoans (phylum Bryozoa), representative colonial animals mostly found in marine environments, some species possess different types of individuals (heterozooids) specialized in different functions such as defense or structural support for their colonies. Among them, the best-known heterozooids are the avicularia, known to function as defenders. The differentiation processes of heterozooids, including avicularia, should be important keys to understand the evolutionary significance of bryozoans. However, the developmental process of avicularium formation remains to be fully understood. In this study, therefore, in order to understand the detailed developmental process and timing of avicularium formation, extensive observations were carried out in a bryozoan species, Bugulina californica (Cheilostomata, Bugulidae), that possesses adventitious avicularia, by performing stereomicroscopy on live materials, in addition to scanning electron microscopy and histological observations. The whole process can be divided into seven stages based on developmental events. Especially notably, at the earlier stages, there are three major budding events that produce proliferating cell masses corresponding to primordial tissues: (1) budding of the peduncle cushion at the outer margin of the distal part of a young autozooid, (2) budding of the head-part primordium from the peduncle cushion, and (3) budding of the polypide inside the head part. Experimental control of temperature showed that 20°C would be the best to maintain B. californica colonies.

Membrane Rafts: Portals for Viral Entry.

Membrane rafts are dynamic, small (10–200 nm) domains enriched with cholesterol and sphingolipids that compartmentalize cellular processes. Rafts participate in roles essential to the lifecycle of different viral families including virus entry, assembly and/or budding events. Rafts seem to participate in virus attachment and recruitment to the cell surface, as well as the endocytic and non-endocytic mechanisms some viruses use to enter host cells. In this review, we will introduce the specific role of rafts in viral entry and define cellular factors implied in the choice of one entry pathway over the others. Finally, we will summarize the most relevant information about raft participation in the entry process of enveloped and non-enveloped viruses.

Dynamic membranes: the multiple roles of P4 and P5 ATPases.

The lipid bilayer of biological membranes has a complex composition, including high chemical heterogeneity, the presence of nanodomains of specific lipids, and asymmetry with respect to lipid composition between the two membrane leaflets. In membrane trafficking, membrane vesicles constantly bud off from one membrane compartment and fuse with another, and both budding and fusion events have been proposed to require membrane lipid asymmetry. One mechanism for generating asymmetry in lipid bilayers involves the action of the P4 ATPase family of lipid flippases; these are biological pumps that use ATP as an energy source to flip lipids from one leaflet to the other. The model plant Arabidopsis (Arabidopsis thaliana) contains 12 P4 ATPases (AMINOPHOSPHOLIPID ATPASE1-12; ALA1-12), many of which are functionally redundant. Studies of P4 ATPase mutants have confirmed the essential physiological functions of these pumps and pleiotropic mutant phenotypes have been observed, as expected when genes required for basal cellular functions are disrupted. For instance, phenotypes associated with ala3 (dwarfism, pollen defects, sensitivity to pathogens and cold, and reduced polar cell growth) can be related to membrane trafficking problems. P5 ATPases are evolutionarily related to P4 ATPases, and may be the counterpart of P4 ATPases in the endoplasmic reticulum. The absence of P4 and P5 ATPases from prokaryotes and their ubiquitous presence in eukaryotes make these biological pumps a defining feature of eukaryotic cells. Here, we review recent advances in the field of plant P4 and P5 ATPases.

Nuclear Egress.

During viral replication, herpesviruses utilize a unique strategy, termed nuclear egress, to translocate capsids from the nucleus into the cytoplasm. This initial budding step transfers a newly formed capsid from within the nucleus, too large to fit through nuclear pores, through the inner nuclear membrane to the perinuclear space. The perinuclear enveloped virion must then fuse with the outer nuclear membrane to be released into the cytoplasm for further maturation, undergoing budding once again at the trans-Golgi network or early endosomes, and ultimately exit the cell non-lytically to spread infection. This first budding process is mediated by two conserved viral proteins, UL31 and UL34, that form a heterodimer called the nuclear egress complex (NEC). This review focuses on what we know about how the NEC mediates capsid transport to the perinuclear space, including steps prior to and after this budding event. Additionally, we discuss the involvement of other viral proteins in this process and how NEC-mediated budding may be regulated during infection.

Engineering mesenchymal stromal/stem cell-derived extracellular vesicles with improved targeting and therapeutic efficiency for the treatment of central nervous system disorders.

Engineering mesenchymal stromal/stem cell-derived extracellular vesicles with improved targeting and therapeutic efficiency for the treatment of central nervous system disorders.

A Rab4‐Regulated Endosomal Compartment Prolongs EGFR Activation in Breast Cancer Cells

Early sorting endosomes are responsible for the proper trafficking of membrane-bound receptors that can drive cancer progression. However, the morphology, cargo sorting and signaling of early endosomes remains poorly understood in breast cancer. We have identified a novel population of enlarged early endosomal vesicles in breast cancer cells and human tumor xenografts (MDAMB231 and T47D) but not in non-cancerous epithelial cells (MCF10A) using confocal and super-resolution 3D dSTORM microscopy. A detailed quantitative analysis of the whole-cell endosomal morphology, cargo sorting, maturation, and regulatory Rab-GTPases associated with this compartment in cancerous breast epithelial cell lines was performed using Airyscan microscopy and 3D rendering approaches. This analysis showed that highly aggressive triple negative MDAMB231 breast cancer cells have larger but significantly fewer EEA1-enriched early sorting endosomes compared to non-cancerous MCF10A cells. Furthermore, live-cell time-lapse imaging showed that, despite normal transferrin recycling, the epidermal growth factor (EGF) and transferrin are found together in these large endosomal vesicles following internalization in MDAMB231 but not in MCF10A cells. Most importantly, these large EEA1-positive MDAMB231 endosomes exhibited prolonged EGF-induced activation of EGFR (p1068). We investigated the role of Rab4-mediated changes on endosome structure and function and found that over-expression of wild-type Rab4 in non-cancerous MCF10A cells produced similar EEA1-positive enlarged early-endosomes with prolonged EGF induced EGFR activation (p1068). Rab4 mutants in dominant-negative or constitutively-active state did not elicit the same effects as the wild-type Rab4, indicating that the on-off cycling of Rab4 is critical for the regulation of endosomal size and number. These large endosomes contain adjacent Rab4 and Rab11 domains, are located primarily at the perinuclear region and can undergo actin-associated budding events to recycle cargo out towards the plasma membrane. Altogether, this extensive characterization of early-endosomes in breast cancer cells has identified a novel Rab4 regulated enlarged endocytic compartment leading to prolonged EGFR activation. Interestingly, our data suggests that a population of “pre-existent” large EEA1-containing endosomes is present in some breast cancer cells, challenging the current views of endosome biogenesis. This research is essential to determining receptor regulation in translational breast cancer models which can impact the optimization of anti-cancer human therapies. Alterations in endocytic regulation in cancer cells may be key to understanding the adaptability of cancer cells and lead to new therapeutic targets. Support or Funding Information This work was supported by the National Institute of Health (NIH) R01 BRG CA20772 Breast cancer cells have altered early-endocytic morphology Representative images from whole-cell Airyscan microscopy assay to quantitatively characterize early-endocytic morphology (with respect to whole-cell morphology) in non-cancerous breast epithelial cells (MCF10A)and breast cancer cell lines (MDAMB231 and T47D) with 3D rendering techniques. Example image from live-cell 4D imaging of endosomal dynamics that facilitates our comprehensive characterization of Rab4-regulated endosomes in breast cancer cells. Prolonged EGF-induced EGFR activation (p1068) in MDAMB231 compared to MCf10A This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single Cell Isolation Using Optical Tweezers.

Optical tweezers offer a non-contact method for selecting single cells and translocating them from one microenvironment to another. We have characterized the optical tweezing of yeast S. cerevisiae and can manipulate single cells at 0.41 ± 0.06 mm/s using a 26.8 ± 0.1 mW from a 785 nm diode laser. We have fabricated and tested three cell isolation devices; a micropipette, a PDMS chip and a laser machined fused silica chip and we have isolated yeast, single bacteria and cyanobacteria cells. The most effective isolation was achieved in PDMS chips, where single yeast cells were grown and observed for 18 h without contamination. The duration of budding in S. cerevisiae was not affected by the laser parameters used, but the time from tweezing until the first budding event began increased with increasing laser energy (laser power × time). Yeast cells tweezed using 25.0 ± 0.1 mW for 1 min were viable after isolation. We have constructed a micro-consortium of yeast cells, and a co-culture of yeast and bacteria, using optical tweezers in combination with the PDMS network of channels and isolation chambers, which may impact on both industrial biotechnology and understanding pathogen dynamics.

Abstract 3994: Anti-metastatic drug MBQ-167 regulates cell polarity via Cdc42

Abstract Cdc42 (cell division control protein 42 homolog) is a Rho GTPase highly conserved among eukaryotes in structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42, aiding cells to polarize and arrange their components accordingly. Its role in cell polarity was first described in budding yeast and subsequently in other eukaryotes, including animal cells determining the direction of motility. Accordingly, in cancer Cdc42 mediates migration, invasion, and spread of tumor cells. Therefore, we target Cdc42 as therapeutic strategy to treat metastatic breast cancer. Our group designed and characterized the small molecule MBQ-167 as a potent inhibitor against Cdc42 activation (IC50=0.1 µM), demonstrating its effectiveness in preventing tumor growth and cell migration in vitro and in vivo. Because in yeast S. cereviciae Cdc42 is well known for regulating the location of budding events, we used this well-characterized model of cell polarization to test our previous findings of MBQ-167 and its specificity for Cdc42. By using a tetracycline-controlled transcriptional inactivation (Tet-Off) system at the promoter region of the CDC42 gene of yeast, we knocked down Cdc42 supplementing the media with doxycycline (DOX; IC=10ug/uL). Next, we analyzed the growth, budding pattern, and Cdc42 activity of budding yeast exposed to DOX, MBQ-167 or in combination. The maximum growth, doubling time and budding polarity of recombinant yeast with the Tet-Off system were reduced when treated with DOX (EC50=10ug/mL), as expected. The same effect was seen in the group treated with MBQ-167 (ED50=100uM), both alone and in combination with DOX. Ultimately, pull-down assays of the active Cdc42-GTP complex confirmed our hypothesis that MBQ-167 disrupts cell polarity through impeding Cdc42 activation. Here we validated MBQ-167 as a Cdc42 inhibitor in another biologic context. and present a method for screening Cdc42 inhibitors and potential therapeutics against metastatic cancer. Citation Format: Michael John Rivera-Robles, Julia Medina-Velázquez, Gabriela M. Asencio-Torres, Sahily González-Crespo, Brian C. Rymond, José Rodríguez-Medina, Suranganie Dharmawardhane. Anti-metastatic drug MBQ-167 regulates cell polarity via Cdc42 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3994.

Yeast cells contain a heterogeneous population of peroxisomes that segregate asymmetrically during cell division.

Here, we used fluorescence microscopy and a peroxisome-targeted tandem fluorescent protein timer to determine the relative age of peroxisomes in yeast. Our data indicate that yeast cells contain a heterogeneous population of relatively old and young peroxisomes. During budding, the peroxisome retention factor inheritance of peroxisomes protein 1 (Inp1) selectively associates to the older organelles, which are retained in the mother cells. Inp2, a protein required for transport of peroxisomes to the bud, preferentially associates to younger organelles. Using a microfluidics device, we demonstrate that the selective segregation of younger peroxisomes to the buds is carefully maintained during multiple budding events. The replicative lifespan of mother cells increased upon deletion of INP2, which resulted in the retention of all organelles in mother cells. These data suggest that, in wild-type yeast, transport of aged and deteriorated peroxisomes to the bud is prevented, whereas the young and vital organelles are preferably transported to the newly forming buds.

Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

Dispersal strategies in the highly polygynous ant Crematogaster (Orthocrema) pygmaea Forel (Formicidae: Myrmicinae).

In ants, dispersal strategies and morphology of female sexuals are generally linked to the mode of colony founding. In species using long-range dispersal tactics, queen/worker dimorphism is generally high and young queens are able to initiate new colonies by themselves, using their metabolic reserves. By contrast, in species using short-range dispersal strategies, queen/worker dimorphism is generally low and, due to their limited metabolic reserves, queens have lost the capacity to raise their brood alone and to found their colony independently. Moreover, polygyny is also often associated with short-range dispersal strategies, although the relationship between the number of queens and the dispersal strategy in ants is not clear-cut. Here, dispersal strategies were investigated in C. pygmaea, a highly polygynous and polydomous ant species from northeastern Brazil. Field observations and laboratory experiments show that this ant exhibits a suite of traits that are more commonly associated with long-range dispersal and independent colony foundation: functional wings in both males and females, high queen/worker dimorphism, strong weight loss in mature queens, nuptial flights and, in the lab, ability of young queens to found new colonies in haplometrotic conditions. On the other hand, this species shows a high degree of polygyny with a strong seasonal component, and, at least under laboratory conditions, mature queens seem able to develop propagules if they are accompanied by at least 10 workers. These features strongly suggest that (1) some of the gynes do not engage in a long-range dispersal but become new queens in their mother colony and (2) that budding events are possible in this species. We therefore speculate that C. pygmaea has a dual dispersal strategy probably related to environmental conditions: some gynes engage in long-range dispersal followed by independent colony foundation at the beginning of rainy season, while others mate in the parental colony and are re-adopted leading to high polygyny. During the rainy season, budding events can lead to colony extension and increased polydomy. Polydomy is commonly thought to improve resource discovery and exploitation through decentralized foraging behavior, a significant advantage during the rainy season when food ressources (mainly floral/extrafloral nectaries and hemipteran honeydew) are more abundant and when colony needs for food supplies are highest.

Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis.

In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.