Characterization of CHMP Polymers at the Mesoscale

Clustering of tetrameric influenza M2 peptides in lipid bilayers investigated by 19F solid-state NMR

The dynamics of nuclear envelope rupture and wound repair are critical biological processes that play essential roles in cell homeostasis. Previous studies on nuclear envelope repair dynamics have mainly focused on small ruptures induced by low-throughput tools such as laser ablation and atomic force microscopy. Recent findings also suggest that nuclear envelope rupture can aid the gene delivery process. We present a device that deterministically porates the cell membrane and nuclear envelope in high throughput, with applications in single-cell studies of wound repair dynamics and in statistical assessments of cell poration. The device consists of sharp nanostructures in arrays of microchannels, enabling precise and localized disruption at both the plasma membrane and nuclear envelope while preserving cell viability. The distribution of endosomal sorting complexes required for transport (ESCRT) proteins after mechanical disruption at both the plasma membrane and nuclear envelope highlights the cell recovery strategy for severe wounds. Interestingly, during this extreme rupture, resources seem to be allocated more towards nuclear envelope repair. The nanostructured microfluidics also facilitate rapid protein expression following naked plasmid DNA transfection, a feat that is only possible through nuclear envelope rupture. High-throughput mechanoporation on membrane systems provides a new perspective on studying complex wound repair dynamics and enables a means to deliver cargo to intracellular and intranuclear domains. This technology opens promising avenues for further research in the field of intracellular delivery and wound healing mechanisms.

Influenza virus assembly and budding

Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.

SummaryIn addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting, and plasma membrane repair [1], the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to seal holes in the reforming nuclear envelope (NE) during mitotic exit [2, 3]. ESCRT-III also acts during interphase to repair the NE upon migration-induced rupture [4, 5], highlighting its key role as an orchestrator of membrane integrity at this organelle. While NE localization of ESCRT-III is dependent upon the ESCRT-III component CHMP7 [3], it is unclear how this complex is able to engage nuclear membranes. Here we show that the N terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the ER, an organelle continuous with the NE, and it provides a platform to direct NE recruitment of ESCRT-III during mitotic exit. CHMP7’s N terminus comprises tandem Winged-Helix domains [6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission.

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication that are involved in both normal processes and pathological conditions. Latent membrane protein 1 (LMP1) is a major viral oncogene that is expressed in most Epstein-Barr virus (EBV)-associated cancers and secreted in EVs. LMP1-modified EVs have the ability to influence recipient cell growth, migration, and differentiation and regulate immune cell function. Despite the significance of LMP1-modified EVs in EBV malignancies, very little is understood about how this protein hijacks the host EV pathway for secretion. Using the biotin identification (BioID) method, we identified LMP1-proximal interacting proteins that are known to play roles in EV formation and protein trafficking. Analysis of the identified LMP1-interacting proteins revealed an enrichment in the ESCRT pathway and associated proteins, including CD63, Syntenin-1, Alix, TSG101, Hrs, and charged multivesicular body proteins (CHMPs). LMP1 transcriptionally upregulated and increased the protein expression of EV biogenesis and secretion genes. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following the knockdown of Syntenin-1, Alix, Hrs, and TSG101, with altered endolysosomal trafficking observed when Syntenin-1 and Hrs expression was reduced. Knockdown of specific ESCRT-III subunits (CHMP4B, -5, and -6) impaired LMP1 packaging and secretion into EVs. Finally, we demonstrate that the efficient secretion of LMP1-modified EVs promotes cell attachment, proliferation, and migration and tumor growth. Together, these results begin to shed light on how LMP1 exploits host ESCRT machinery to direct the incorporation of the viral oncoprotein into the EV pathway for secretion to alter the tumor microenvironment.IMPORTANCE LMP1 is a notable viral protein that contributes to the modification of EV content and tumor microenvironment remodeling. LMP1-modified EVs enhance tumor proliferation, migration, and invasion potential and promote radioresistance. Currently, the mechanisms surrounding LMP1 incorporation into the host EV pathways are not well understood. This study revealed that LMP1 utilizes Hrs, Syntenin-1, and specific components of the ESCRT-III complex for release from the cell, enhancement of EV production, and metastatic properties of cancer cells. These findings begin to unravel the mechanism of LMP1 EV trafficking and may provide new targets to control EBV-associated cancers.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

The effect of delta-9-tetrahydrocannabinol (delta-9-THC) on the growth kinetics and morphology of rat B103 neuroblastoma cells was assessed. Delta-9-THC in doses ranging from 10(-4) to 10(-7) M inhibited cellular growth in a dose-dependent fashion as evidenced by delay in doubling time, decrease in saturation density, and decrease in efficiency of plating. The inhibition in cellular growth was paralleled by dose-related alterations in cell morphology. Modifications included rounding of cells, retraction of neurites, blebbing of the cell surface, and exfoliation of the plasma membrane. Cytoplasmic alterations included distension of the endoplasmic reticulum, Golgi apparatus, and perinuclear space, and macrovacuolization. Intracytoplasmic laminated inclusions and vesicular clusters were suggestive of membrane repair in drug-treated cells. These morphological changes were accompanied by cytoskeletal rearrangement in the absence of significant alteration in the concentration of total cytoskeletal protein. Autoradiographic examination of the intracellular fate of 3H-delta-9-THC demonstrated that the drug was confined to the cytoplasmic compartment and often associated with macrovacuoles. These results suggest that delta-9-THC interacts with cellular membranes, thereby altering neuroblastoma cell growth and behavior.

Impact of radiation therapy on the oncolytic adenovirus dl520: Implications on the treatment of glioblastoma

Simian virus 40 (SV40) appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection to aid in virus dissemination. To investigate the contribution of VP4 to cell lysis, VP4 was expressed in mammalian cells where it was predominantly observed along the nuclear periphery. The integrity of the nuclear envelope was compromised in these cells, resulting in the mislocalization of a soluble nuclear marker. Using assays that involved the cellular expression of VP4 or the treatment of cells with purified VP4, we found that the central hydrophobic domain and a proximal C-terminal nuclear localization signal of VP4 were required for (i) cytolysis associated with prolonged expression; (ii) nuclear envelope accumulation; and (iii) disruption of the nuclear, red blood cell, or host cell membranes. Furthermore, a conserved proline within the hydrophobic domain was required for membrane perforation, suggesting that this residue was crucial for VP4 cytolytic activity. These results indicate that VP4 forms pores in the nuclear membrane leading to lysis and virus release.

L–Arabinose-based coordination polymers were synthesized by the oxidation of L–arabinose using warm HNO3 to form arabinaric acid and allowing the resulting arabinaric acid to react with the different metal ions such as zinc(II), lead(II), copper(II), iron(III), and chromium(III). Only zinc(II) and lead(II) ions produced crystalline coordination polymers suitable for analysis while copper(II) produced a gummy solid. Preliminary characterization of the coordination polymers was based on their solubility, melting points, UV–Visible spectra, and infrared spectra. The coordination polymers were insoluble in common organic solvents and in acidic aqueous solutions but soluble in moderately basic aqueous solutions (pH 12). The zinc(II) and lead(II) coordination polymers have melting points above 300°C while the copper(II) polymer melts from 218–225°C. UV–Visible spectra showed that these polymers have maximum absorption in the ultraviolet region. Zinc(II), lead(II), and copper(II) arabinarate coordination polymers had maximum absorbance at λmax = 217 nm, λmax = 240 nm, and λmax = 220 nm, respectively. The infrared spectra of the coordination polymers indicated the association of the arabinarate ligand with the metal ions. Introduction and incorporation of molecular species such as azobenzene and molecular iodine to the coordination polymers were also investigated. Infrared spectra obtained from the azobenzene inclusion experiments showed additional infrared absorption bands below 1000 cm –1 implying absorption of azobenzene within the polymers. Iodometric titrations also confirmed the inclusion of iodine in the coordination polymers. The potential of the coordination polymers to incorporate lead(II) ions in aqueous solutions was also investigated using column elution of different sample concentrations of lead(II). Atomic Absorption Spectrometric analysis illustrated significant lead reduction of up to 80.70 ppm of lead(II) on the eluted samples. Introduction Rapid industrialization in the modern world today causes severe pollution in the environment. This may be due to toxic, inorganic, or non-biodegradable materials released to the environment as waste products during industrial processes. Heavy metals, like lead, are among the most harmful of the environmental elemental pollutants. Lead contamination from excessive exposure has been found to cause several health hazards and long term detrimental effects. Prominent conventional processes for the removal of lead from aqueous mixtures, such as sludge separation, chemical precipitation, and electrochemical treatments are relatively expensive and can cause secondary pollutants (Manahan, 1993 as cited in Acabal et al., 2000). Due to the problems of pollution, “green” industrial productions of compounds and their derivatives based on renewable starting materials and biodegradable products are developed (Kiely, 2001). Carbohydrates are biodegradable, widely available, and generally economical. Hence, their study for chemical and industrial applications would be very sensible. Because of their ease in functionalization forming sugar derivatives, they could serve as valuable building blocks for potentially useful polymers (Abrahams et al., 2003). Heavy metal sequestering properties of certain types of these polymers provide an alternative and potentially economical treatment process for heavy metal removal and recovery.

Ultrastructural studies of the envelopment and release of guinea pig herpes-like virus in cultured cells

Nuclear transport proteins such as CSE1, NUP93 and Importinα have recently been shown to be chromatin-associated proteins in yeast, which have unexpected functions in gene regulation. Here we report interactions between the mammalian histone acetyltransferase CBP with nuclear transport proteins CAS (a CSE1 homologue) and Importin-α (Impα) and NUP93. CAS was found to bind the SRC1 interaction domain (SID) of CBP via a leucine-rich motif in the N-terminus of the protein, that is conserved in other SID-binding proteins. Co-immunoprecipitation experiments also revealed that CBP and Impα proteins form a complex. As Impα is a known acetylation target of CBP/p300, and is recycled to the cytoplasm via the exportin CAS, we investigated whether HDAC inhibitors would alter the subcellular localisation of these proteins. Treatment of COS-1 cells with the HDAC inhibitors trichostatin A or sodium butyrate resulted in sequestration of Impα in the nuclear envelope, accumulation of CAS in nuclear aggregates, and an increased number of CBP-containing PML bodies per cell. In addition, HDACi treatment appeared to enhance the association of Impα and CBP in co-immunoprecipitation experiments. Our results provide evidence for novel functional interactions between the chromatin modification enzyme CBP and nuclear transport proteins in mammalian cells.

The nuclear envelope (NE) undergoes dynamic remodeling to maintain NE integrity, a process involving the inner nuclear membrane protein LEM2 recruiting CHMP7/Cmp7 and then ESCRT-III. However, prior work has hinted at CHMP7/ESCRT-independent mechanisms. To identify such mechanisms, we studied NE assembly in Schizosaccharomyces japonicus, a fission yeast that undergoes partial mitotic NE breakdown and reassembly. S. japonicus cells lacking Cmp7 have compromised NE sealing after mitosis but are viable. A genetic screen identified mutations that promote NE integrity in cmp7Δ cells. Unexpectedly, loss of Lem2 or its interacting partner Nur1 suppressed cmp7Δ defects. In the absence of Cmp7, Lem2 formed aggregates that appear to interfere with ESCRT-independent NE sealing. A gain-of-function mutation implicated a membrane and ESCRT-III regulator, Alx1, in this alternate pathway. Additional results suggest a potentially general role for unsaturated fatty acids in NE integrity. These findings establish the existence of mechanisms for NE sealing independent of the canonical ESCRT pathway.