Biology subjects

Funaya, C.

Publications and source records attributed to Funaya, C..

9 recordsLinked to original sources

bioRxiv2026

ZDHHC17–Mediated Palmitoylation of Hepatitis E Virus ORF3 Protein Regulates Vectorial Trafficking in Polarized Epithelial Cells

The hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide and is transmitted enterically along the gut-liver axis. Epithelial cell polarity in the gut and liver plays a critical role in HEV transmission. In intestinal epithelial cells, HEV enters through the apical membrane and is released basolaterally to access the bloodstream, whereas in hepatocytes, the virus enters basolaterally and is secreted apically into the bile duct. In this study, we sought to identify the viral and host determinants governing directional HEV secretion in both tissue types. Using polarized intestinal and hepatocyte models, we found that the small phosphoprotein ORF3, which is essential for progeny secretion, localizes predominantly to the apical membrane, in contrast to the ORF2 capsid protein. We further identified the palmitoyltransferase ZDHHC17 as a specific ORF3 interactor that mediates its apical localization and promotes HEV progeny release. Using automated cell segmentation and quantitative image analysis, we screened a panel of ORF3 mutants and found that positively charged residues within the N-terminus regulate membrane association. In addition, we identified a conserved PIFIQP motif that mediates the critical interaction with the ankyrin repeat domain (ARD) of ZDHHC17. Leveraging this interaction, we generated high-confidence structural models of ORF3 in complex with the ZDHHC17 ARD using AlphaFold. These models, further validated by molecular dynamics simulations, revealed additional ORF3 residues involved in the interaction. Collectively, our findings define key mechanisms underlying directional HEV release and provide broader insights into trafficking processes in polarized epithelial cells.

Cell Biology↗

bioRxiv2024

Morphology-dependent entry kinetics and spread of influenza A virus

Influenza A viruses (IAV) display a broad variety of morphologies ranging from spherical to long filamentous virus particles. These diverse phenotypes are believed to allow the virus to overcome various immunological and pulmonary barriers during entry into the airway epithelium and influence the viral entry pathway. Remarkably, lab-adapted IAV strains lost this morphological variance and exhibit preferred spherical morphology. However, it remains unclear which factors lead to this lab-adapted preference and which pulmonary defense factors are responsible for the preferred filamentous morphology in physiological settings. In this study, we established fluorescent reporter viruses with spherical or filamentous morphology but with the same surface glycoproteins. We developed a correlative fluorescence and scanning electron microscopy workflow to analyze the impact of viral morphology on cell-to-cell spread and identify conditions under which IAV with either spherical or filamentous morphology confer an advantage. Our findings demonstrate that filamentous IAV cell-to-cell spread is significantly slower in various cell lines, which can explain the predominant spherical morphology in lab-adapted strains. This observation is consistent with delayed entry kinetics of filamentous viruses structurally analyzed by cellular cryo-electron tomography. We found that cellular junction integrity and mucin do not exert morphology-dependent inhibition of IAV cell-to-cell spread. On the other hand, filamentous virions confer an advantage under the pressure exerted by neutralizing antibodies against hemagglutinin.

microbiology↗

bioRxiv2023

Microtubule inner proteins of Plasmodium are essential for transmission of malaria parasites

Microtubule inner proteins, MIPs, are microtubule associated proteins that bind to tubulin from the luminal side. MIPs can be found in axonemes to stabilize flagellar beat or within cytoplasmic microtubules. Plasmodium spp. are the causative agents of malaria that feature different forms across a complex life cycle with both unique and divergent microtubule-based arrays. Here we investigate the role of four MIPs in a rodent malaria parasite for their role in transmission to and from the mosquito. We show by single and double gene deletions that SPM1 and TrxL1, MIPs associated with the subpellicular microtubules are dispensable for transmission from the vertebrate host to the mosquito and back. In contrast, FAP20 and FAP52, MIPs associated with the axonemes of gametes, are essential for transmission to mosquitoes but only if both genes are deleted. In the absence of both, FAP20 and FAP52 the B-tubule of the axoneme partly detaches from the A-tubule resulting in the deficiency of axonemal beating and hence gamete formation and egress. Our data suggest that a high level of redundancy ensures microtubule stability in the transmissive stages of Plasmodium, which is important for parasite transmission.

microbiology↗

bioRxiv2022

Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, ApoE-LPs transport from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and extracellular vesicles (EVs) along the late endosomal trafficking route. Moreover, complexes of ApoE-LPs and CD63-positive EVs were found to be transmitted from cell to cell. Given the important role of ApoE in viral infections, we studied the hepatitis C virus (HCV) and found that the viral replicase protein NS5A is enriched in ApoE-containing intraluminal vesicles. Interaction between NS5A and ApoE is required for the efficient release of EVs containing viral RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders. Author SummaryThe post-Golgi egress pathway of hepatocyte-derived ApoE-containing lipoproteins (ApoE-LPs) is largely unknown. By using integrative imaging analyses, we show that ApoE-LPs are enriched in CD63-positive endosomes suggesting that these endosomes might be a central hub for the storage of ApoE-LPs from which they are released into the circulation. In addition, we provide evidence for the co-egress of ApoE-LPs with extracellular vesicles (EVs) along the late endosomal route and their transfer from cell to cell. This pathway is hijacked by the hepatitis C virus that induces the production of ApoE-associated EVs containing viral RNA. Given the important role of ApoE in multiple metabolic, degenerative and infectious diseases, and the role of EVs in cell-to-cell communication, these results provide important information how perturbations of ApoE might contribute to various pathophysiologies.

microbiology↗

bioRxiv2021

Malaria transmission relies on concavin-mediated maintenance of Plasmodium sporozoite cell shape

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here we report on a newly identified protein, concavin, that is important for maintenance of the sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localized at the cytoplasmic periphery of sporozoites and concavin(-) sporozoites progressively rounded up upon entry of salivary glands. These rounded concavin(-) sporozoites failed to pass through the narrow salivary ducts and were hence rarely ejected by mosquitoes. However, normally shaped concavin(-) sporozoites could be transmitted and migrated in the skin or skin like environments. Strikingly, motile concavin(-) sporozoites could disintegrate while migrating through narrow strictures in the skin leading to parasite arrest or death and decreased transmission efficiency. We suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. SIGNIFICANCEMalaria parasites are transmitted by Anopheles mosquitoes and rely on rapid migration for establishing an infection. We identified and characterized a protein, named concavin, essential for maintaining the shape of the sporozoite. Concavin is a membrane associated protein facing the cytoplasm suggesting that it contributes to riveting the plasma membrane to the subtending inner membrane complex. Sporozoites lacking concavin can round up in the salivary glands, are less well transmitted to mice and disintegrate while migrating in the skin. Hence, concavin is essential for parasite transmission and infectivity. Highlights- A membrane associated protein is essential for Plasmodium shape maintenance -Migrating parasites disintegrate in the absence of concavin -First protein essential for cellular integrity of Plasmodium sporozoites -Thickened and deformed Plasmodium sporozoites fail to be transmitted by mosquitoes

microbiology↗

bioRxiv2021

Asynchronous nuclear cycles in multinucleated Plasmodium falciparum enable rapid proliferation

Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2n progression and each proliferative cycle produces a heterogeneous number of progeny. Here, by tracking nuclei and DNA replication, we show that individual nuclei replicate their DNA at different times, despite residing in a shared cytoplasm. Extrapolating from experimental data using mathematical modeling, we demonstrate that a limiting factor must exist that slows down the nuclear multiplication rate. Indeed, our data show that temporally overlapping DNA replication events were significantly slower than partially or non-overlapping events. Our findings suggest an evolutionary pressure that selects for asynchronous DNA replication, balancing available resources with rapid pathogen proliferation.

microbiology↗

bioRxiv2021

An extended DNA-free intranuclear compartment organizes centrosomal microtubules in Plasmodium falciparum

Rapid proliferation of Plasmodium falciparum parasites in human red blood cells is the cause of malaria and is underpinned by an unconventional cell division mode, called schizogony. Contrary to model organisms, P. falciparum replicates by multiple rounds of closed and asynchronous nuclear divisions that are not interrupted by cytokinesis. Organization and dynamics of the critical nuclear division factors are, however, poorly understood. Centriolar plaques, the centrosomes of P. falciparum, are important regulators of division and serve as microtubule organizing centers. Early microscopy studies reveal an acentriolar, amorphous structure although its detailed organization remains elusive. Intranuclear microtubules mediate chromosome segregation, but the small size of parasite nuclei has precluded detailed analysis of their arrangement by classical fluorescence microscopy. We apply recently developed STED, expansion microscopy, and live cell imaging protocols to describe the reconfiguration of microtubules during schizogony. Analysis of centrin, nuclear pore, and microtubule positioning reveals a bipartite organization of the centriolar plaque. While centrin is extranuclear, we confirm by correlative light and electron tomography that microtubules are nucleated in a previously unknown and extended intranuclear compartment, which is devoid of chromatin. This study enables us to build a working model of the organization of an unconventional centrosome and better understand the diversity of eukaryotic cell division modes.

cell biology↗

bioRxiv2019

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and ESCRT proteins as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that ESCRT proteins directly function in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

cell biology↗

bioRxiv2017

Cell-specific modulation of nuclear pore complexes controls cell cycle entry during asymmetric division

The acquisition of cellular identity is coupled to changes in the nuclear periphery and nuclear pore complexes (NPCs). Whether and how these changes determine cell fate remains unclear. We have uncovered a mechanism regulating NPC acetylation to direct cell fate after asymmetric division in budding yeast. The lysine deacetylase Hos3 associates specifically with daughter cell NPCs during mitosis to delay cell cycle entry (Start). Hos3-dependent deacetylation of nuclear basket and central channel nucleoporins establishes daughter cell-specific nuclear accumulation of the transcriptional repressor Whi5 during anaphase and perinuclear silencing of the CLN2 gene in the following G1 phase. Hos3-dependent coordination of both events restrains Start in daughter but not in mother cells. We propose that deacetylation modulates transport-dependent and - independent functions of NPCs, leading to differential cell cycle progression in mother and daughter cells. Similar mechanisms might regulate NPC functions in specific cell types and/or cell cycle stages in multicellular organisms.

cell biology↗