De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.

Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding.

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination.

A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling.

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

Dynamics of Translation of Single mRNA Molecules In Vivo.

Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5' UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.

Publisher Correction: The cytoplasmic dynein transport machinery and its many cargoes.

In Figure 1c of the original article, ARP1 was incorrectly labelled as ARP11. The highlight for reference 37 was mistakenly placed under reference 36 and the highlight for reference 29 should have also referred to reference 16 (instead of 19). The HTML and PDF versions of the article have now been corrected.

The cytoplasmic dynein transport machinery and its many cargoes.

Cytoplasmic dynein 1 is an important microtubule-based motor in many eukaryotic cells. Dynein has critical roles both in interphase and during cell division. Here, we focus on interphase cargoes of dynein, which include membrane-bound organelles, RNAs, protein complexes and viruses. A central challenge in the field is to understand how a single motor can transport such a diverse array of cargoes and how this process is regulated. The molecular basis by which each cargo is linked to dynein and its cofactor dynactin has started to emerge. Of particular importance for this process is a set of coiled-coil proteins - activating adaptors - that both recruit dynein-dynactin to their cargoes and activate dynein motility.

CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis.

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.

A protein-tagging system for signal amplification in gene expression and fluorescence imaging.

Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.

Allosteric communication in the dynein motor domain.

Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. Here, we report X-ray and electron microscopy (EM) structures of yeast dynein bound to different ATP analogs, which collectively provide insight into the roles of dynein's two major ATPase sites, AAA1 and AAA3, in the conformational change mechanism. ATP binding to AAA1 triggers a cascade of conformational changes that propagate to all six AAA domains and cause a large movement of the "linker," dynein's mechanical element. In contrast to the role of AAA1 in driving motility, nucleotide transitions in AAA3 gate the transmission of conformational changes between AAA1 and the linker, suggesting that AAA3 acts as a regulatory switch. Further structural and mutational studies also uncover a role for the linker in regulating the catalytic cycle of AAA1. Together, these results reveal how dynein's two major ATP-binding sites initiate and modulate conformational changes in the motor domain during motility.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2.

The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.

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Cortical dynein controls microtubule dynamics to generate pulling forces that position microtubule asters.

Dynein at the cortex contributes to microtubule-based positioning processes such as spindle positioning during embryonic cell division and centrosome positioning during fibroblast migration. To investigate how cortical dynein interacts with microtubule ends to generate force and how this functional association impacts positioning, we have reconstituted the 'cortical' interaction between dynein and dynamic microtubule ends in an in vitro system using microfabricated barriers. We show that barrier-attached dynein captures microtubule ends, inhibits growth, and triggers microtubule catastrophes, thereby controlling microtubule length. The subsequent interaction with shrinking microtubule ends generates pulling forces up to several pN. By combining experiments in microchambers with a theoretical description of aster mechanics, we show that dynein-mediated pulling forces lead to the reliable centering of microtubule asters in simple confining geometries. Our results demonstrate the intrinsic ability of cortical microtubule-dynein interactions to regulate microtubule dynamics and drive positioning processes in living cells.

Repeat-associated non-AUG translation induces cytoplasmic aggregation of CAG repeat-containing RNAs.

CAG trinucleotide repeat expansions cause several neurodegenerative diseases, including Huntington's disease and spinocerebellar ataxia. RNAs with expanded CAG repeats contribute to disease in two unusual ways. First, these repeat-containing RNAs may agglomerate in the nucleus as foci that sequester several RNA-binding proteins. Second, these RNAs may undergo aberrant repeat-associated non-AUG (RAN) translation in multiple frames and produce aggregation-prone proteins. The relationship between RAN translation and RNA foci, and their relative contributions to cellular dysfunction, are unclear. Here, we show that CAG repeat-containing RNAs that undergo RAN translation first accumulate at nuclear foci and, over time, are exported to the cytoplasm. In the cytoplasm, these RNAs are initially dispersed but, upon RAN translation, aggregate with the RAN translation products. These RNA-RAN protein agglomerates sequester various RNA-binding proteins and are associated with the disruption of nucleocytoplasmic transport and cell death. In contrast, RNA accumulation at nuclear foci alone does not produce discernable defects in nucleocytoplasmic transport or cell viability. Inhibition of RAN translation prevents cytoplasmic RNA aggregation and alleviates cell toxicity. Our findings demonstrate that RAN translation-induced RNA-protein aggregation correlates with the key pathological hallmarks observed in disease and suggest that cytoplasmic RNA aggregation may be an underappreciated phenomenon in CAG trinucleotide repeat expansion disorders.

Patronin regulates the microtubule network by protecting microtubule minus ends.

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR).

The chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, nor actin remodeling following CAR activation. However, CAR T cells still require LAT for an optimal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.

Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility.

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation.

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.