Regulation of Tubulin dynamics by the +Tip tracking protein Mal3

The Microtubule (MT) network is a central component of the eukaryotic cell cytoskeleton. In the fission yeast S. pombe, a complex of three proteins specifically tracks MT +ends and stabilizes MTs in the cell. It is composed of the proteins Mal3, Tip1 and Tea2. Mal3, the S. pombe homologue of EB1, is a highly conserved ubiquitous protein found to be at the centre of many MT related processes. Tip1 is a CLIP170 homologue and Tea2 a kinesin-like motor protein. The mechanism by which they target the growing end of MTs and stabilize them is still unknown. A combination of biochemistry, electron microscopy and crystallography were used in an attempt to get a more precise understanding of the MT stabilization by this +Tip complex. Protein-A pull-down of the endogenous complex and analysis of its constituents by mass spectrometry revealed that Tea2 and Tip1 form a tight stoichiometric complex, making a much more labile interaction with Mal3. Biochemical experiments, light scattering and DIC microscopy demonstrate that Mal3 stabilizes the MT structure in a stoichiometric fashion by suppressing catastrophe events. 3D helical reconstruction of electron micrographs of Mal3 bound to the MT show that it most probably stabilizes the MT structure by bridging protofilaments together. Deletion mutant analysis suggests that contact with one of the protofilaments is via an interaction between the charged tails of tubulin and Mal3. Mal3 MT binding domain structure was solved by X-ray crystallography so that eventually it may be docked into a higher resolution electron microscopy map to provide a more precise structural insight on how Mal3 stabilizes the MT lattice. The EM analysis also shows that Mal3 regulates MT structure in vitro by restraining their protofilament number to 13, which is the number always found in vivo, and by driving the assembly of MTs with a high proportion of A-lattice. It is the first time that a protein is found to promote formation of A-lattice MTs. The fact that EB1 is such a ubiquitous protein reopens the question of MT structure in cells and has important implications for in vivo MT dynamics.Rob Cros

Engineered ferritin for lanthanide binding

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295–375 nm, fluorescence excitation

Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells

Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ

Visualization of translation reorganization upon persistent collision stress in mammalian cells

UNLABELLED: Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress. Here we visualize the effect of a persistent collision stress on translation using in situ cryo electron tomography. We observe that low dose anisomycin collision stress leads to the stabilization of Z-site bound tRNA on elongating 80S ribosomes, as well as to the accumulation of an off-pathway 80S complex possibly resulting from collision splitting events. We visualize collided disomes in situ , occurring on compressed polysomes and revealing a stabilized geometry involving the Z-tRNA and L1 stalk on the stalled ribosome, and eEF2 bound to its collided rotated-2 neighbor. In addition, non-functional post-splitting 60S complexes accumulate in the stressed cells, indicating a limiting Ribosome associated Quality Control clearing rate. Finally, we observe the apparition of tRNA-bound aberrant 40S complexes shifting with the stress timepoint, suggesting a succession of different initiation inhibition mechanisms over time. Altogether, our work visualizes the changes of translation complexes under persistent collision stress in mammalian cells, indicating how perturbations in initiation, elongation and quality control processes contribute to an overall reduced protein synthesis. SUMMARY: Using in situ cryo electron tomography we visualized the reorganization of mammalian translation processes during a persistent collision stress

Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells

International audienceAberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ

Humanized archaeal ferritin as a tool for cell targeted delivery

Human ferritins have been extensively studied to be used as nanocarriers for diverse applications and could represent a convenient alternative for targeted delivery of anticancer drugs and imaging agents. However, the most relevant limitation to their applications is the need for highly acidic experimental conditions during the initial steps of particle/cargo assembly, a process that could affect both drug stability and the complete reassembly of the ferritin cage. To overcome this issue the unique assembly of Archaeoglobus fulgidus ferritin was genetically engineered by changing a surface exposed loop of 12 amino acids connecting B and C helices to mimic the sequence of the analogous human H-chain ferritin loop. This new chimeric protein was shown to maintain the unique, cation linked, association–dissociation properties of Archaeoglobus fulgidus ferritin occurring at neutral pH values, while exhibiting the typical human H-homopolymer recognition by the transferrin receptor TfR1. The chimeric protein was confirmed to be actively and specifically internalized by HeLa cells, thus representing a unique nanotechnological tool for cell-targeted delivery of possible payloads for diagnostic or therapeutic purposes. Moreover, it was demonstrated that the 12 amino acids’ loop is necessary and sufficient for binding to the transferrin receptor. The three-dimensional structure of the humanized Archaeoglobus ferritin has been obtained both as crystals by X-ray diffraction and in solution by cryo-EM

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Intermediates in SARS-CoV-2 spike-mediated cell entry

SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors

Discovery of neutralizing SARS-CoV-2 antibodies enriched in a unique antigen specific B cell cluster.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery