Ribosome Distribution in HeLa Cells during the Cell Cycle

In this study, we employed a surface-specific antibody against the large ribosome subunit to investigate the distribution of ribosomes in cells during the cell cycle. The antibody, anti-L7n, was raised against an expansion segment (ES) peptide from the large subunit ribosomal protein L7, and its ribosome-surface specificity was evident from the positive immuno-reactivity of ribosome particles and the detection of 60 S immune-complex formation by an immuno-electron microscopy. Using immunofluorescent staining, we have microscopically revealed that ribosomes are dispersed in the cytoplasm of cells throughout all phases of the cell cycle, except at the G2 phase where ribosomes show a tendency to gather toward the nuclear envelope. The finding in G2 cells was confirmed by electron microscopy using a morphometric assay and paired t test. Furthermore, further observations have shown that ribosomes are not distributed immune-fluorescently with nuclear envelope markers including the nuclear pore complex, the integral membrane protein gp210, the inner membrane protein lamin B2, and the endoplasm reticulum membrane during cell division we propose that the mechanism associated with ribosome segregation into daughter cells could be independent of the processes of disassembly and reassembly of the nuclear envelope

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain

Curr. Biol.

Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport [1-3]. Dynactin can bind to vesicles and liposomes containing acidic phospholipids [4], but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes [5]. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion [6]. In particular, Rab27a tethers melanosomes to the actin cytoskeleton [7-9]. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule- dependent transport pathways through the Golgi and from endosomes to the Golgi [10-16]. We demonstrate that dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes

The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes

Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport [1-3]. Dynactin can bind to vesicles and liposomes containing acidic phospholipids [4], but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes [5]. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion [6]. In particular, Rab27a tethers melanosomes to the actin cytoskeleton [7-9]. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule- dependent transport pathways through the Golgi and from endosomes to the Golgi [10-16]. We demonstrate that dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. Phase separation is driven, in part, by hydrophobic and electrostatic interactions. While the N protein forms spherical assemblies with unstructured RNA, it forms asymmetric condensates with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle

MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii

This work was supported by the National Institutes of Health (AI139201 and AI137767 to GEW, each including salary support; GM141743 to DMW, including salary support; F31AI145214 to RVS, including predoctoral fellowship stipend support; and T32AI055402 to GEW, including predoctoral fellowship stipend support for AKS). The work was also supported by the Canadian Institutes of Health Research (148596 to MJB), the Canada Research Chair program (to MJB, salary support) and the American Heart Association (20POST35220017 to RSK, including postdoctoral fellowship stipend support).Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.Publisher PDFPeer reviewe