Structural determination and modeling of ciliary microtubules.

The axoneme, a microtubule-based array at the center of every cilium, has been the subject of structural investigations for decades, but only recent advances in cryo-EM and cryo-ET have allowed a molecular-level interpretation of the entire complex to be achieved. The unique properties of the nine doublet microtubules and central pair of singlet microtubules that form the axoneme, including the highly decorated tubulin lattice and the docking of massive axonemal complexes, provide opportunities and challenges for sample preparation, 3D reconstruction and atomic modeling. Here, the approaches used for cryo-EM and cryo-ET of axonemes are reviewed, while highlighting the unique opportunities provided by the latest generation of AI-guided tools that are transforming structural biology.

Three structural solutions for bacterial adhesion pilus stability and superelasticity.

Bacterial adhesion pili are key virulence factors that mediate host-pathogen interactions in diverse epithelial environments. Deploying a multimodal approach, we probed the structural basis underpinning the biophysical properties of pili originating from enterotoxigenic (ETEC) and uropathogenic bacteria. Using cryo-electron microscopy we solved the structures of three vaccine target pili from ETEC bacteria, CFA/I, CS17, and CS20. Pairing these and previous pilus structures with force spectroscopy and steered molecular dynamics simulations, we find a strong correlation between subunit-subunit interaction energies and the force required for pilus unwinding, irrespective of genetic similarity. Pili integrate three structural solutions for stabilizing their assemblies: layer-to-layer interactions, N-terminal interactions to distant subunits, and extended loop interactions from adjacent subunits. Tuning of these structural solutions alters the biophysical properties of pili and promotes the superelastic behavior that is essential for sustained bacterial attachment.

Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin.

Following binding to the thin filament, ß-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular contraction. However, key features of the cardiac-specific actin-myosin interaction remain uncertain, including the structural effect of ADP release from myosin, which is rate-limiting during force generation. In fact, ADP release slows under experimental load or in the intact heart due to the afterload, thereby adjusting cardiac muscle power output to meet physiological demands. To further elucidate the structural basis of this fundamental process, we used a combination of cryo-EM reconstruction methodologies to determine structures of the human cardiac actin-myosin-tropomyosin filament complex at better than 3.4 Å-resolution in the presence and in the absence of Mg2+·ADP. Focused refinements of the myosin motor head and its essential light chains in these reconstructions reveal that small changes in the nucleotide-binding site are coupled to significant rigid body movements of the myosin converter domain and a 16-degree lever arm swing. Our structures provide a mechanistic framework to understand the effect of ADP binding and release on human cardiac ß-myosin, and offer insights into the force-sensing mechanism displayed by the cardiac myosin motor.

Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation.

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct, where the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366-372. Cryo-EM analysis of fully decorated actin-tropomyosin filaments with wild-type and mutant S1, yielded 3.4-3.6 Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other when filaments were decorated with either mutant or wild-type S1. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of filaments containing the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the high potential energy associated with tropomyosin positioning in wild-type filaments favors an effective transition to B- and C-states following release of myosin from the thin filaments during relaxation.

Dynamic interaction network involving the conserved intrinsically disordered regions in human eIF5.

Translation initiation in eukaryotes requires multiple eukaryotic translation initiation factors (eIFs) and involves continuous remodeling of the ribosomal preinitiation complex (PIC). The GTPase eIF2 brings the initiator Met-tRNAi to the PIC. Upon start codon selection and GTP hydrolysis, promoted by eIF5, eIF2-GDP is released in complex with eIF5. Here, we report that two intrinsically disordered regions (IDRs) in eIF5, the DWEAR motif and the C-terminal tail (CTT) dynamically contact the folded C-terminal domain (CTD) and compete with each other. The eIF5-CTD•CTT interaction favors eIF2ß binding to eIF5-CTD, whereas the eIF5-CTD•DWEAR interaction favors eIF1A binding, which suggests how intramolecular contact rearrangement could play a role in PIC remodeling. We show that eIF5 phosphorylation by CK2, which is known to stimulate translation and cell proliferation, significantly increases the eIF5 affinity for eIF2. Our results also indicate that the eIF2ß subunit has at least two, and likely three eIF5-binding sites.

The Central Role of the F-Actin Surface in Myosin Force Generation.

Actin is one of the most abundant and versatile proteins in eukaryotic cells. As discussed in many contributions to this Special Issue, its transition from a monomeric G-actin to a filamentous F-actin form plays a critical role in a variety of cellular processes, including control of cell shape and cell motility. Once polymerized from G-actin, F-actin forms the central core of muscle-thin filaments and acts as molecular tracks for myosin-based motor activity. The ATP-dependent cross-bridge cycle of myosin attachment and detachment drives the sliding of myosin thick filaments past thin filaments in muscle and the translocation of cargo in somatic cells. The variation in actin function is dependent on the variation in muscle and non-muscle myosin isoform behavior as well as interactions with a plethora of additional actin-binding proteins. Extensive work has been devoted to defining the kinetics of actin-based force generation powered by the ATPase activity of myosin. In addition, over the past decade, cryo-electron microscopy has revealed the atomic-evel details of the binding of myosin isoforms on the F-actin surface. Most accounts of the structural interactions between myosin and actin are described from the perspective of the myosin molecule. Here, we discuss myosin-binding to actin as viewed from the actin surface. We then describe conserved structural features of actin required for the binding of all or most myosin isoforms while also noting specific interactions unique to myosin isoforms.

Cryo-EM and Molecular Docking Shows Myosin Loop 4 Contacts Actin and Tropomyosin on Thin Filaments.

The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding of nonmuscle cell motility. Indeed, experimental approaches such as fiber diffraction, cryoelectron microscopy, and three-dimensional reconstruction have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryogenic electron microscopy reconstruction of myosin-S1-decorated F-actin-tropomyosin together with atomic scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle ß-myosin complexes; masseter myosin, which shares sequence identity with ß-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39-42 residue-long tropomyosin pseudorepeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation.

Does changing the reference frame affect infant categorization of the spatial relation BETWEEN?

Past research has shown that variation in the target objects depicting a given spatial relation disrupts the formation of a category representation for that relation. In the current research, we asked whether changing the orientation of the referent frame depicting the spatial relation would also disrupt the formation of a category representation for that relation. Experiments 1 to 3 provided evidence that 6- and 7-month-olds formed a category representation for BETWEEN when a diamond shape was depicted in different locations between two vertical or horizontal reference bars during familiarization and in a novel location between the same orientation of bars during test. By contrast, in Experiment 4, same-age infants did not form a category representation for BETWEEN when the diamond shape was depicted between two vertical (or horizontal) bars during familiarization and between two horizontal (or vertical) bars during test. Moreover, in Experiment 5, 9- and 10-month-olds did form a category representation for BETWEEN when the orientation of the referent bars depicting the relation changed from familiarization to test. The findings suggest that the formation of category representations for spatial relations by infants is affected by changes to either target (figure) or referent (ground).

The role of visual attention in multiple object tracking: evidence from ERPs.

We examined the role of visual attention in the multiple object tracking (MOT) task by measuring the amplitude of the N1 component of the event-related potential (ERP) to probe flashes presented on targets, distractors, or empty background areas. We found evidence that visual attention enhances targets and suppresses distractors (Experiment 1 & 3). However, we also found that when tracking load was light (two targets and two distractors), accurate tracking could be carried out without any apparent contribution from the visual attention system (Experiment 2). Our results suggest that attentional selection during MOT is flexibly determined by task demands as well as tracking load and that visual attention may not always be necessary for accurate tracking.

Neural markers of subordinate-level categorization in 6- to 7-month-old infants.

Subordinate-level category-learning processes in infants were investigated with ERP and looking-time measures. ERPs were recorded while 6- to 7-month-olds were presented with Saint Bernard images during familiarization, followed by novel Saint Bernards interspersed with Beagles during test. In addition, infant looking times were measured during a paired-preference test (novel Saint Bernard vs. novel Beagle) conducted at the conclusion of ERP recording. Slow wave activity corresponded with learning a familiarized category at the subordinate and basic levels, whereas Negative central (Nc) and P400 components were linked with novel category preference. The results provide the first evidence identifying the neural markers of subordinate-level categorization observed in looking-time tasks conducted with infants. Moreover, when considered in conjunction with prior research investigating the neural markers of basic-level categorization in infants, the findings indicate that (1) slow wave and Nc components of infant ERP waveforms are general markers for processes of category learning on the one hand and novel category preference on the other, (2) novel category preference for a contrast category at the basic and subordinate levels have the Nc component in common, but novel category preference at the subordinate level is accompanied by an additional P400 component, a finding in keeping with the notion that subordinate-level categorization is governed by mechanisms supplementary to those underlying basic-level categorization, and (3) slow wave activity associated with subordinate-level learning followed that associated with basic-level learning by approximately 200 ms, a result in accord with a coarse-to-fine scheme for the emergence of category partitioning.

Time course of visual attention in infant categorization of cats versus dogs: evidence for a head bias as revealed through eye tracking.

Previous looking time studies have shown that infants use the heads of cat and dog images to form category representations for these animal classes. The present research used an eye-tracking procedure to determine the time course of attention to the head and whether it reflects a preexisting bias or online learning. Six- to 7-month-olds were familiarized with cats or dogs in upright or inverted orientations and then tested with a novel cat and novel dog in the same orientation. In the upright orientation, infants fixated head over body throughout familiarization; with inversion, no head preference was observed. These findings suggest that infant reliance on the head to categorize cats versus dogs results from a bias that pushes attention to the head.