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Max is an obligate dimerization partner for the Myc transcription factors and for several repressors, such as Mnt, Mxd1-4 and Mga, collectively thought to antagonize Myc function in transcription and oncogenesis. Mga, in particular, is part of the variant Polycomb group repressive complex PRC1.6. Here, we show that ablation of the distinct PRC1.6 subunit Pcgf6 – but not Mga – accelerates Myc-induced lymphomagenesis in Eµ-myc transgenic mice. Unexpectedly, however, Pcgf6 loss shows no significant impact on transcriptional profiles, in neither pre-tumoral B-cells, nor lymphomas. Altogether, these data unravel an unforeseen, Mga- and PRC1.6-independent tumor suppressor activity of Pcgf6.

During tissue morphogenesis, the changes in cell shape, resulting from cell-generated forces, often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. In this study, we identify an actomyosin-dependent mechanism involving Rab11-mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures (“Rab11 vesicles”) by promoting a directional bias in dynein-mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network, and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding.

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5WT and an ATPase-deficient mutant, ScMreB5E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane.

Neuronal extracellular vesicles (EVs) are locally released from presynaptic terminals, carrying cargoes critical for intercellular signaling and disease. EVs are derived from endosomes, but it is unknown how these cargoes are directed to the EV pathway rather than for conventional endolysosomal degradation. Here, we find that endocytic machinery plays an unexpected role in maintaining a release-competent pool of EV cargoes at synapses. Endocytic mutants, including nervous wreck (nwk), shibire/dynamin, and AP-2, unexpectedly exhibit local presynaptic depletion specifically of EV cargoes. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo synaptotagmin-4 (Syt4) and suppress lethality upon overexpression of the EV cargo amyloid precursor protein (APP). These EV defects are genetically separable from canonical endocytic functions in synaptic vesicle recycling and synaptic growth. Endocytic machinery opposes the endosomal retromer complex to regulate EV cargo levels and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that locally promotes cargo loading into synaptic EVs.

Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)–rich lamellipodia that normally drive directional migration. Using live-cell TIRF microscopy, we demonstrated that ATG9A-positive vesicles are targeted toward the migration front of polarized cells, where their exocytosis correlates with protrusive activity. Finally, we found that ATG9A was critical for efficient delivery of β1 integrin to the leading edge and normal adhesion dynamics. Collectively, our data uncover a new function for ATG9A protein and indicate that ATG9A-positive vesicles are mobilized during chemotactic stimulation to facilitate expansion of the lamellipodium and its anchorage to the extracellular matrix.

In this review, Braschi et al. provide an updated consensus nomenclature for components of dynein motors and their assembly factors., Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.

Myosin filament–based regulation supplements actin filament–based regulation to control the strength and speed of contraction in heart muscle. In diastole, myosin motors form a folded helical array that inhibits actin interaction; during contraction, they are released from that array. A similar structural transition has been observed in mammalian skeletal muscle, in which cooling below physiological temperature has been shown to reproduce some of the structural features of the activation of myosin filaments during active contraction. Here, we used small-angle x-ray diffraction to characterize the structural changes in the myosin filaments associated with cooling of resting and relaxed trabeculae from the right ventricle of rat hearts from 39°C to 7°C. In intact quiescent trabeculae, cooling disrupted the folded helical conformation of the myosin motors and induced extension of the filament backbone, as observed in the transition from diastole to peak systolic force at 27°C. Demembranation of trabeculae in relaxing conditions induced expansion of the filament lattice, but the structure of the myosin filaments was mostly preserved at 39°C. Cooling of relaxed demembranated trabeculae induced changes in motor conformation and filament structure similar to those observed in intact quiescent trabeculae. Osmotic compression of the filament lattice to restore its spacing to that of intact trabeculae at 39°C stabilized the helical folded state against disruption by cooling. The myosin filament structure and motor conformation of intact trabeculae at 39°C were largely preserved in demembranated trabeculae at 27°C or above in the presence of Dextran, allowing the physiological mechanisms of myosin filament–based regulation to be studied in those conditions.

Condensin I is a five-subunit protein complex that is central to mitotic chromosome assembly in eukaryotic cells. Despite recent progress, its molecular mechanisms of action remain to be fully elucidated. By using Xenopus egg extracts as a functional assay, we find that condensin I complexes harboring mutations in its kleisin subunit CAP-H produce chromosomes with confined axes in the presence of topoisomerase IIα (topo IIα) and highly compact structures (termed “beans”) with condensin-positive central cores in its absence. The bean phenotype depends on the SMC ATPase cycle and can be reversed by subsequent addition of topo IIα. The HEAT repeat subunit CAP-D2, but not CAP-G, is essential for the bean formation. Notably, loop extrusion activities of the mutant complexes cannot explain the chromosomal defects they exhibit in Xenopus egg extracts, implying that a loop extrusion–independent mechanism contributes to condensin I–mediated chromosome assembly and shaping. We provide evidence that condensin–condensin interactions underlie these processes.

α- and β-tubulins are encoded by multigene families, but the role of tubulin diversity for microtubule function has been a longstanding mystery. A new study (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010155) shows that the two budding yeast α-tubulins have distinct roles during mitotic spindle positioning.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2–Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

X-ray diffraction studies of muscle have been tremendously powerful in providing fundamental insights into the structures of, for example, the myosin and actin filaments in a variety of muscles and the physiology of the cross-bridge mechanism during the contractile cycle. However, interpretation of x-ray diffraction patterns is far from trivial, and if modeling of the observed diffraction intensities is required it needs to be performed carefully with full knowledge of the possible pitfalls. Here, we discuss (1) how x-ray diffraction can be used as a tool to monitor various specific muscle properties and (2) how to get the most out of the rest of the observed muscle x-ray diffraction patterns by modeling where the reliability of the modeling conclusions can be objectively tested. In other x-ray diffraction methods, such as protein crystallography, the reliability of every step of the process is estimated and quoted in published papers. In this way, the quality of the structure determination can be properly assessed. To be honest with ourselves in the muscle field, we need to do as near to the same as we can, within the limitations of the techniques that we are using. We discuss how this can be done. We also use test cases to reveal the dos and don’ts of using x-ray diffraction to study muscle physiology.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder caused primarily by mutations to thick and thinfilament proteins. Although thin filament mutations are less prevalent than their oft-studied thick filament counterparts, they are frequently associated with severe patient phenotypes and can offer important insight into fundamental disease mechanisms. We have performed a detailed study of tropomyosin (TPM1) E192K, a variant of uncertain significance associated with HCM. Molecular dynamics revealed that E192K results in a more flexible TPM1 molecule, which could affect its ability to regulate crossbridges. In vitro motility assays of regulated actin filaments containing TPM1 E192K showed an overall loss of Ca2+ sensitivity. To understand these effects, we used multiscale computational models that suggested a subtle phenotype in which E192K leads to an inability to completely inhibit actin–myosin crossbridge activity at low Ca2+. To assess the physiological impact of the mutation, we generated patient-derived engineered heart tissues expressing E192K. These tissues showed disease features similar to those of the patients, including cellular hypertrophy, hypercontractility, and diastolic dysfunction. We hypothesized that excess residual crossbridge activity could be triggering cellular hypertrophy, even if the overall Ca2+ sensitivity was reduced by E192K. To test this hypothesis, the cardiac myosin–specific inhibitor mavacamten was applied to patient-derived engineered heart tissues for 4 d followed by 24 h of washout. Chronic mavacamten treatment abolished contractile differences between control and TPM1 E192K engineered heart tissues and reversed hypertrophy in cardiomyocytes. These results suggest that the TPM1 E192K mutation triggers cardiomyocyte hypertrophy by permitting excess residual crossbridge activity. These studies also provide direct evidence that myosin inhibition by mavacamten can counteract the hypertrophic effects of mutant tropomyosin.

Cowell et al. show that talin rod domain–containing protein 1 (TLNRD1), a protein with homology to the central region of the integrin regulator, talin, has retained the diverse interactions of talin R7R8 but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly., Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain–containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif–containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.

Olson and Machesky discuss work from the Way laboratory that reveals how ARP3B isoform-specific ARP2/3 complexes lead to faster disassembly of branched actin filaments., The ARP2/3 complex promotes branched actin networks, but the importance of specific subunit isoforms is unclear. In this issue, Galloni, Carra, et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202102043) show that MICAL2 mediates methionine oxidation of ARP3B, thus destabilizing ARP2/3 complexes and leading to disassembly of branched actin filaments.

Tseng et al. show the Bro1 V domain stimulates Vps4 to promote ESCRT-III–driven intralumenal vesicle formation. This mode of stimulation is required for coordinated cargo incorporation during ESCRT-mediated multivesicular body biogenesis., Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

Passive force generated in titin plays an important role in maintaining sarcomere structure. In this paper, van der Pijl et al. describe a mechanism by which muscle ankyrin repeat protein 1 (MARP1) locks titin to the thin filament to increase passive force., Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin–N2A element, found in titin’s molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin–N2A. Mechanics and super-resolution microscopy revealed that MARP1 “locks” titin–N2A to the sarcomeric thin filament, causing increased extension of titin’s elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

Huang et al. reveal that the microcephaly-related protein WDR62 possesses an intrinsic affinity for curved microtubules and regulates spindle dynamics by functioning as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin., WDR62 is a microcephaly-related, microtubule (MT)-associated protein (MAP) that localizes to the spindle pole and regulates spindle organization, but the underlying mechanisms remain elusive. Here, we show that WDR62 regulates spindle dynamics by recruiting katanin to the spindle pole and further reveal a TPX2–Aurora A–WDR62–katanin axis in cells. By combining cellular and in vitro experiments, we demonstrate that WDR62 shows preference for curved segments of dynamic GDP-MTs, as well as GMPCPP- and paclitaxel-stabilized MTs, suggesting that it recognizes extended MT lattice. Consistent with this property, WDR62 alone is inefficient in recruiting katanin to GDP-MTs, while WDR62 complexed with TPX2/Aurora A can potently promote katanin-mediated severing of GDP-MTs in vitro. In addition, the MT-binding affinity of WDR62 is autoinhibited through JNK phosphorylation-induced intramolecular interaction. We propose that WDR62 is an atypical MAP and functions as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin to orchestrate the regulation of spindle dynamics.

Interferon responses in the nasopharyngeal epithelium are critical to prevent severe COVID-19. Epithelial cells alone are unable to stop SARS-CoV-2 growth, but sufficient early interferon can be provided by non-epithelial cells or a co-infecting virus. Autoantibodies block early interferon, enhancing the risk of severe COVID-19., Interferons establish innate antiviral immunity. Two recent papers in JEM by Lopez et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20211211) and Cheemarla et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20210583) show that an appropriate supply of antiviral interferon enables epithelial cells of the nasopharyngeal mucosa to inhibit SARS-CoV-2 growth and that interferon-induced mucosal genes serve as biomarkers of infection.

Single-cell profiling demonstrates multifarious dysregulation of innate immune phenotype associated with COVID-19 severity. Severe COVID-19 is associated with hyperactivation of neutrophils and NK cells, while monocytes take on tolerogenic phenotypes. Meanwhile, mild COVID-19 is associated with limited, or rapidly resolved, immune perturbation., Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-κB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity–associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention., Graphical Abstract

Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca2+-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca2+. Altogether, these effects outline a motor isoform-specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.

The local rules that control cell shape and movement are not understood. Here, Pipathsouk et al. discover a novel nanoscale organization of a key actin regulator that could explain how cellular protrusions expand and self-straighten and how transcellular holes are repaired., How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.

SUMO homeostasis promotes error-free chromosome segregation. Quan et al. report the structure of a targeting peptide of the Ulp2 desumoylase bound to yeast Ctf3/CENP-I. Disrupting the interaction produces hyper-sumoylated kinetochores, demonstrating the existence of a conserved and dedicated pathway for the regulation of kinetochore sumoylation., The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle–regulated processes.

Galloni, Carra, et al. demonstrate that Arp3B isoform–specific Arp2/3 complexes generate branched actin networks with faster disassembly kinetics. This increased turnover is due to oxidation of Met293 of Arp3B by the methionine monooxygenase MICAL2, which is recruited to the branched actin network by coronin 1C., The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.

The thick (myosin-containing) filaments of vertebrate skeletal muscle are arranged in a hexagonal lattice, interleaved with an array of thin (actin-containing) filaments with which they interact to produce contraction. X-ray diffraction and electron microscopy have shown that there are two types of thick filament lattice. In the simple lattice, all filaments have the same orientation about their long axis, while in the super lattice, nearest neighbors have rotations differing by 60°. Tetrapods (amphibians, reptiles, birds, mammals) typically have only a super lattice, while the simple lattice is confined to fish. We have carried out X-ray diffraction and electron microscopy of the soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat and found that while the EDL has a super-lattice, as expected, the SOL has a simple lattice. The EDL and SOL of the rat are unusual in being essentially pure fast and slow muscles respectively. The mixed fiber content of most tetrapod muscles and/or lattice disorder may explain why the simple lattice has not been apparent in these vertebrates before. This is supported by only weak simple lattice diffraction in the X-ray pattern of mouse SOL, which has a greater mix of fiber types than rat. We conclude that the simple lattice might be common in tetrapods. The correlation between fiber type and filament lattice arrangement suggests that the lattice arrangement may contribute to the functional properties of a muscle.SummaryThe three-dimensional arrangement of thick filaments in skeletal muscle is studied by X-ray diffraction and electron microscopy. A correlation is found between thick filament lattice type (simple or super lattice) and fiber type (fast/slow). This suggests that lattice organization contributes to muscle functional properties

Genetic studies revealed that Abl family kinases interact functionally with microtubules, but the mechanism by which Abl kinases regulate microtubules remains unclear. Hu et al. provide the first evidence that the Abl family kinase Abl2 directly binds microtubules to regulate microtubule dynamics., Abl family kinases are essential regulators of cell shape and movement. Genetic studies revealed functional interactions between Abl kinases and microtubules (MTs), but the mechanism by which Abl family kinases regulate MTs remains unclear. Here, we report that Abl2 directly binds to MTs and regulates MT behaviors. Abl2 uses its C-terminal half to bind MTs, an interaction mediated in part through electrostatic binding to tubulin C-terminal tails. Using purified proteins, we found that Abl2 binds growing MTs and promotes MT polymerization and stability. In cells, knockout of Abl2 significantly impairs MT growth, and this defect can be rescued via reexpression of Abl2. Stable reexpression of an Abl2 fragment containing the MT-binding domain alone was sufficient to restore MT growth at the cell edge. These results show Abl2 uses its C-terminal half to bind MTs and directly regulate MT dynamics.

Baumgart et al. measure the amount of dimeric and polymeric tubulin at mitotic centrosomes in C. elegans by light and electron microscopy. Centrosomes concentrate soluble tubulin tenfold over the cytoplasm, suggesting that centrosomal microtubule nucleation may be driven in part by concentrating tubulin., During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained by a lack of knowledge of the amount of soluble and polymeric tubulin at mitotic centrosomes. Here we combined light microscopy and serial-section electron tomography to measure the amount of dimeric and polymeric tubulin at mitotic centrosomes in early C. elegans embryos. We show that a C. elegans one-cell stage centrosome at metaphase contains >10,000 microtubules with a total polymer concentration of 230 µM. Centrosomes concentrate soluble α/β tubulin by about 10-fold over the cytoplasm, reaching peak values of 470 µM, giving a combined total monomer and polymer tubulin concentration at centrosomes of up to 660 µM. These findings support in vitro data suggesting that microtubule nucleation in C. elegans centrosomes is driven in part by concentrating soluble tubulin.

Wang et al. identify Rab7 as a novel regulator of primary cilia disassembly. Their findings demonstrate that Rab7 localization to primary cilia is required for intraciliary F-actin polymerization, which is indispensable for the regulation of cilia ectocytosis and disassembly., The primary cilium is a sensory organelle that protrudes from the cell surface. Primary cilia undergo dynamic transitions between assembly and disassembly to exert their function in cell signaling. In this study, we identify the small GTPase Rab7 as a novel regulator of cilia disassembly. Depletion of Rab7 potently induced spontaneous ciliogenesis in proliferating cells and promoted cilia elongation during quiescence. Moreover, Rab7 performs an essential role in cilia disassembly; knockdown of Rab7 blocked serum-induced ciliary resorption, and active Rab7 was required for this process. Further, we demonstrate that Rab7 depletion significantly suppresses cilia tip excision, referred to as cilia ectocytosis, which has been identified as required for cilia disassembly. Mechanically, the failure of F-actin polymerization at the site of excision of cilia tips caused suppression of cilia ectocytosis on Rab7 depletion. Overall, our results suggest a novel function for Rab7 in regulating cilia ectocytosis and cilia disassembly via control of intraciliary F-actin polymerization.

Puleo et al. identify the Ena/VASP protein EVL as a crucial actin polymerization factor at focal adhesions, which regulates focal adhesion maturation and mechanosensing. Interestingly, they show that EVL promotes mechanically directed motility and durotactic invasion yet suppresses chemotactic response., The mechanical properties of a cell’s microenvironment influence many aspects of cellular behavior, including cell migration. Durotaxis, the migration toward increasing matrix stiffness, has been implicated in processes ranging from development to cancer. During durotaxis, mechanical stimulation by matrix rigidity leads to directed migration. Studies suggest that cells sense mechanical stimuli, or mechanosense, through the acto-myosin cytoskeleton at focal adhesions (FAs); however, FA actin cytoskeletal remodeling and its role in mechanosensing are not fully understood. Here, we show that the Ena/VASP family member, Ena/VASP-like (EVL), polymerizes actin at FAs, which promotes cell-matrix adhesion and mechanosensing. Importantly, we show that EVL regulates mechanically directed motility, and that suppression of EVL expression impedes 3D durotactic invasion. We propose a model in which EVL-mediated actin polymerization at FAs promotes mechanosensing and durotaxis by maturing, and thus reinforcing, FAs. These findings establish dynamic FA actin polymerization as a central aspect of mechanosensing and identify EVL as a crucial regulator of this process.

Roy et al. highlight a harmful cross-talk that can arise between spindle assembly checkpoint silencing and chromosome biorientation due to the involvement of protein phosphatase 1 in both the processes., Accurate chromosome segregation during cell division requires the spindle assembly checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect kinetochore–microtubule attachments. While the SAC and error correction are both regulated by protein phosphatase 1 (PP1), which silences the SAC and stabilizes kinetochore–microtubule attachments, how these distinct PP1 functions are coordinated remains unclear. Here, we investigate the contribution of PP1, docked on its conserved kinetochore receptor Spc105/Knl1, to SAC silencing and attachment regulation. We find that Spc105-bound PP1 is critical for SAC silencing but dispensable for error correction; in fact, reduced PP1 docking on Spc105 improved chromosome segregation and viability of mutant/stressed states. We additionally show that artificially recruiting PP1 to Spc105/Knl1 before, but not after, chromosome biorientation interfered with error correction. These observations lead us to propose that recruitment of PP1 to Spc105/Knl1 is carefully regulated to ensure that chromosome biorientation precedes SAC silencing, thereby ensuring accurate chromosome segregation.

The cell cortex is populated by various proteins, but it is unclear how they interact to change cell shape. Jiang and Harris find that the kinase Par-1 is required for Diaphanous-based actin bundles, and that these bundles intersperse with separately induced Arp2/3 networks to form an actin cap that grows into a metaphase compartment of the syncytial Drosophila embryo., Cell structure depends on the cortex, a thin network of actin polymers and additional proteins underlying the plasma membrane. The cell polarity kinase Par-1 is required for cells to form following syncytial Drosophila embryo development. This requirement stems from Par-1 promoting cortical actin caps that grow into dome-like metaphase compartments for dividing syncytial nuclei. We find the actin caps to be a composite material of Diaphanous (Dia)-based actin bundles interspersed with independently formed, Arp2/3-based actin puncta. Par-1 and Dia colocalize along extended regions of the bundles, and both are required for the bundles and for each other’s bundle-like localization, consistent with an actin-dependent self-reinforcement mechanism. Par-1 helps establish or maintain these bundles in a cortical domain with relatively low levels of the canonical formin activator Rho1-GTP. Arp2/3 is required for displacing the bundles away from each other and toward the cap circumference, suggesting interactions between these cytoskeletal components could contribute to the growth of the cap into a metaphase compartment.

Palani et al. reveal a new mechanism by which the F-actin binding protein tropomyosin is regulated. They find that phosphorylation of tropomyosin reduces its affinity for F-actin, allowing the competing Adf1 to bind and sever actin filaments., Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. In muscle cells, tropomyosin is subject to calcium regulation, but its regulation in nonmuscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin-severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of nonmuscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.

The active force of mammalian skeletal muscle is reduced at low temperatures. Caremani et al. reveal that this is due to the rise of a population of myosin motors captured in a refractory state insensitive to muscle activation., Myosin motors in the thick filament of resting striated (skeletal and cardiac) muscle are trapped in an OFF state, in which the motors are packed in helical tracks on the filament surface, inhibiting their interactions with actin and utilization of ATP. To investigate the structural changes induced in the thick filament of mammalian skeletal muscle by changes in temperature, we collected x-ray diffraction patterns from the fast skeletal muscle extensor digitorum longus of the mouse in the temperature range from near physiological (35°C) to 10°C, in which the maximal isometric force (T0) shows a threefold decrease. In resting muscle, x-ray reflections signaling the OFF state of the thick filament indicate that cooling produces a progressive disruption of the OFF state with motors moving away from the ordered helical tracks on the surface of the thick filament. We find that the number of myosin motors in the OFF state at 10°C is half of that at 35°C. At T0, changes in the x-ray signals that report the fraction and conformation of actin-attached motors can be explained if the threefold decrease in force associated with lowering temperature is due not only to a decrease in the force-generating transition in the actin-attached motors but also to a twofold decrease in the number of such motors. Thus, lowering the temperature reduces to the same extent the fraction of motors in the OFF state at rest and the fraction of motors attached to actin at T0, suggesting that motors that leave the OFF state accumulate in a disordered refractory state that makes them unavailable for interaction with actin upon stimulation. This regulatory effect of temperature on the thick filament of mammalian skeletal muscle could represent an energetically convenient mechanism for hibernating animals.

Kindlin-3 regulates podosome stability by recruiting leupaxin to podosomes, which in turn controls PTP-PEST activity and paxillin phosphorylation. Kindlin-3 deficiency allows formation of initial adhesion patches containing talin, vinculin, and paxillin, whereas paxillin family proteins are dispensable for podosome formation., Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling. How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we report that kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. We demonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation, requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes is kindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3–null cells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or any paxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes but also regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylation and downstream signaling.

Phosphorylation of kinetochore proteins destabilizes improper kinetochore–microtubule attachments. Asai et al. find that SET/TAF1, an inhibitor of the PP2A phosphatase, binds shugoshin 2 and corrects erroneous kinetochore–microtubule attachment by maintaining Aurora B kinase activity. Therefore, SET has a key role in establishing chromosome bi-orientation by balancing Aurora B and PP2A activity., The accurate regulation of phosphorylation at the kinetochore is essential for establishing chromosome bi-orientation. Phosphorylation of kinetochore proteins by the Aurora B kinase destabilizes improper kinetochore–microtubule attachments, whereas the phosphatase PP2A has a counteracting role. Imbalanced phosphoregulation leads to error-prone chromosome segregation and aneuploidy, a hallmark of cancer cells. However, little is known about the molecular events that control the balance of phosphorylation at the kinetochore. Here, we show that localization of SET/TAF1, an oncogene product, to centromeres maintains Aurora B kinase activity by inhibiting PP2A, thereby correcting erroneous kinetochore–microtubule attachment. SET localizes at the inner centromere by interacting directly with shugoshin 2, with SET levels declining at increased distances between kinetochore pairs, leading to establishment of chromosome bi-orientation. Moreover, SET overexpression induces chromosomal instability by disrupting kinetochore–microtubule attachment. Thus, our findings reveal the novel role of SET in fine-tuning the phosphorylation level at the kinetochore by balancing the activities of Aurora B and PP2A.

In mitosis, the outer kinetochore is assembled to signal the spindle assembly checkpoint and to attach chromosomes to spindle microtubules. Bonner et al. demonstrate that outer kinetochore assembly requires the enrichment of Aurora B kinase at the inner kinetochore by the conserved central region of INCENP., Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using Xenopus laevis egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates., Graphical Abstract

Takahashi et al. perform a genome-wide CRISPR screen using the HaloTag-LC3 assay to gain insight into the mechanisms of phagophore closure. They identify a role for VPS37A in coordinating the ESCRT assembly on the phagophore for membrane closure., The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

Abad et al. report that the chromosomal passenger complex (CPC), an essential regulator of error-free chromosome segregation, is targeted to chromosomes via Borealin-mediated direct interaction with nucleosomes. This is critical for subsequent histone modification–dependent centromere enrichment and function of the CPC during metaphase., Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin–nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.

Azoitei, Noh, et al. engineer fluorescent biosensors to measure activation at the subcellular level and with subsecond kinetics of GEF-H1, a Rho GTPase that regulates cytoskeletal dynamics. In combination with computational image time series analysis, the biosensors reveal the synergistic role of microtubule dynamics and Src phosphorylation in regulating GEF-H1 activity locally during cell migration., Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0–2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.

Chen et al. reconstitute endocytosis in a cell-free system and show that cargo sorting requires the dynamic dissociation of clathrin during the growth phase of the clathrin-coated pit formation., Clathrin-mediated endocytosis depends on the formation of functional clathrin-coated pits that recruit cargos and mediate the uptake of those cargos into the cell. However, it remains unclear whether the cargos in the growing clathrin-coated pits are actively monitored by the coat assembly machinery. Using a cell-free reconstitution system, we report that clathrin coat formation and cargo sorting can be uncoupled, indicating that a checkpoint is required for functional cargo incorporation. We demonstrate that the ATPase Hsc70 and a dynamic exchange of clathrin during assembly are required for this checkpoint. In the absence of Hsc70 function, clathrin assembles into pits but fails to enrich cargo. Using single-molecule imaging, we further show that uncoating takes place throughout the lifetime of the growing clathrin-coated pits. Our results suggest that the dynamic exchange of clathrin, at the cost of the reduced overall assembly rates, primarily serves as a proofreading mechanism for quality control of endocytosis.

Cardiac troponin I Ser44 is a downstream target for protein kinase C. The current studies show heightened phosphorylation develops during contractile dysfunction in failing human myocardium and in rat models of pressure overload and aging with contractile dysfunction., Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future.

Retromer sorts diverse membrane proteins into recycling tubules/vesicles from the endosome. How it precisely selects its cargos is not well described. Suzuki et al. reveal that retromer interacts with a bipartite sorting signal, which facilitates precise cargo recognition., Retromer is an evolutionarily conserved protein complex, which sorts functionally diverse membrane proteins into recycling tubules/vesicles from the endosome. Many of the identified cargos possess a recycling signal sequence defined as ØX[L/M/V], where Ø is F/Y/W. However, this sequence is present in almost all proteins encoded in the genome. Also, several identified recycling sequences do not follow this rule. How then does retromer precisely select its cargos? Here, we reveal that an additional motif is also required for cargo retrieval. The two distinct motifs form a bipartite recycling signal recognized by the retromer subunits, Vps26 and Vps35. Strikingly, Vps26 utilizes different binding sites depending on the cargo, allowing retromer to recycle different membrane proteins. Thus, retromer interacts with cargos in a more complex manner than previously thought, which facilitates precise cargo recognition.

Intracellular transport can be driven by unidirectional motors acting in opposing directions, but how bidirectional transport of cargo is regulated is unclear. Kendrick et al. show that the dynein-activating adaptor Hook3 interacts with the opposite-polarity motors cytoplasmic dynein-1 and the kinesin KIF1C. In vitro, Hook3 can scaffold both motors for bidirectional motility on microtubules., The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the “tail” of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3’s ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors.

In this work, Kumar et al. use their previously developed talin tension sensor to study the immediate response of cells to uniaxial stretch. Tension measurements together with high-resolution electron microscopy reveal a novel role for the actin cross-linking protein filamin A in mediating tensional symmetry within the F-actin network., Cell sensing of externally applied mechanical strain through integrin-mediated adhesions is critical in development and physiology of muscle, lung, tendon, and arteries, among others. We examined the effects of strain on force transmission through the essential cytoskeletal linker talin. Using a fluorescence-based talin tension sensor (TS), we found that uniaxial stretch of cells on elastic substrates increased tension on talin, which was unexpectedly independent of the orientation of the focal adhesions relative to the direction of strain. High-resolution electron microscopy of the actin cytoskeleton revealed that stress fibers (SFs) are integrated into an isotropic network of cortical actin filaments in which filamin A (FlnA) localizes preferentially to points of intersection between SFs and cortical actin. Knockdown (KD) of FlnA resulted in more isolated, less integrated SFs. After FlnA KD, tension on talin was polarized in the direction of stretch, while FlnA reexpression restored tensional symmetry. These data demonstrate that a FlnA-dependent cortical actin network distributes applied forces over the entire cytoskeleton–matrix interface.

Our study reveals Drg1 as a new binding partner of Dishevelled. The Drg1–Dishevelled association regulates Daam1 and RhoA interactions and activity, leading to polymerization and stability of the actin cytoskeleton, a process that is essential for proper multiciliation., Cilia are critical for proper embryonic development and maintaining homeostasis. Although extensively studied, there are still significant gaps regarding the proteins involved in regulating ciliogenesis. Using the Xenopus laevis embryo, we show that Dishevelled (Dvl), a key Wnt signaling scaffold that is critical to proper ciliogenesis, interacts with Drg1 (developmentally regulated GTP-binding protein 1). The loss of Drg1 or disruption of the interaction with Dvl reduces the length and number of cilia and displays defects in basal body migration and docking to the apical surface of multiciliated cells (MCCs). Moreover, Drg1 morphants display abnormal rotational polarity of basal bodies and a decrease in apical actin and RhoA activity that can be attributed to disruption of the protein complex between Dvl and Daam1, as well as between Daam1 and RhoA. These results support the concept that the Drg1–Dvl interaction regulates apical actin polymerization and stability in MCCs. Thus, Drg1 is a newly identified partner of Dvl in regulating ciliogenesis.

Schrank and Gautier discuss the generation and function of nuclear domains during DNA repair with a special focus on nuclear actin polymerization., Eukaryotic nuclei are organized into nuclear domains that unite loci sharing a common function. These domains are essential for diverse processes including (1) the formation of topologically associated domains (TADs) that coordinate replication and transcription, (2) the formation of specialized transcription and splicing factories, and (3) the clustering of DNA double-strand breaks (DSBs), which concentrates damaged DNA for repair. The generation of nuclear domains requires forces that are beginning to be identified. In the case of DNA DSBs, DNA movement and clustering are driven by actin filament nucleators. Furthermore, RNAs and low-complexity protein domains such as RNA-binding proteins also accumulate around sites of transcription and repair. The link between liquid–liquid phase separation and actin nucleation in the formation of nuclear domains is still unknown. This review discusses DSB repair domain formation as a model for functional nuclear domains in other genomic contexts.

Microtubule bundles in the spindle midzone have been reported to either promote or hinder chromosome movement. Pamula et al. examine the assembly dynamics of midzone microtubule bundles during anaphase and how chromosome segregation is impacted by aberrant bundle assembly., In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.

High-resolution structural information is invaluable for understanding muscle function. Savich et al. use bifunctional spin labeling to determine the orientation of the myosin lever arm in muscle fibers at high resolution under ambient conditions, augmenting previous insights obtained from fluorescence and EM., Despite advances in x-ray crystallography, cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain (LCD; lever arm) under ambient conditions in vertebrate muscle. Here, we measure the orientation of LCD elements in demembranated muscle fibers by electron paramagnetic resonance (EPR) using a bifunctional spin label (BSL) with an angular resolution of 4°. To achieve stereoselective site-directed labeling with BSL, we engineered a pair of cysteines in the myosin regulatory light chain (RLC), either on helix E or helix B, which are roughly parallel or perpendicular to the myosin lever arm, respectively. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtain EPR spectra from which the angular distributions of BSL, and thus the lever arm, can be determined with high resolution relative to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibits both ordered (σ ∼9–11°) and disordered (σ > 38°) populations. Using these angles to determine the orientation of the lever arm (LCD combined with converter subdomain), we observe that the oriented population corresponds to a lever arm that is perpendicular to the muscle fiber axis and that the addition of ATP in the absence of Ca2+ (inducing relaxation) shifts the orientation to a much more disordered orientational distribution. Although the detected orientation of the myosin light chain lever arm is ∼33° different than predicted from a standard “lever arm down” model based on cryo-EM of actin decorated with isolated myosin heads, it is compatible with, and thus augments and clarifies, fluorescence polarization, x-ray interference, and EM data obtained from muscle fibers. These results establish feasibility for high-resolution detection of myosin LCD rotation during muscle contraction.

Synergism between myosin II contractility and cofilin activity modulates serotonin-dependent axon growth. Normally, cofilin-dependent decreases in actin density are compensated by increases in point contact density and traction force; however, myosin hyperactivation leads to catastrophic decreases in actin network density and neurite retraction., Serotonin (5-HT) is known to increase the rate of growth cone advance via cofilin-dependent increases in retrograde actin network flow and nonmuscle myosin II activity. We report that myosin II activity is regulated by PKC during 5-HT responses and that PKC activity is necessary for increases in traction force normally associated with these growth responses. 5-HT simultaneously induces cofilin-dependent decreases in actin network density and PKC-dependent increases in point contact density. These reciprocal effects facilitate increases in traction force production in domains exhibiting decreased actin network density. Interestingly, when PKC activity was up-regulated, 5-HT treatments resulted in myosin II hyperactivation accompanied by catastrophic cofilin-dependent decreases in actin filament density, sudden decreases in traction force, and neurite retraction. These results reveal a synergistic relationship between cofilin and myosin II that is spatiotemporally regulated in the growth cone via mechanocatalytic effects to modulate neurite growth.

Using chemical genetics, we show that acute inhibition of otherwise rapamycin-insensitive TORC2 triggers a slow increase in plasma membrane tension that provokes snapping of the bonds between adaptor proteins and polymerizing actin filaments and ultimately the cessation of endocytosis in yeast., Target of rapamycin complex 2 (TORC2) is a conserved protein kinase that regulates multiple plasma membrane (PM)–related processes, including endocytosis. Direct, chemical inhibition of TORC2 arrests endocytosis but with kinetics that is relatively slow and therefore inconsistent with signaling being mediated solely through simple phosphorylation cascades. Here, we show that in addition to and independently from regulation of the phosphorylation of endocytic proteins, TORC2 also controls endocytosis by modulating PM tension. Elevated PM tension, upon TORC2 inhibition, impinges on endocytosis at two different levels by (1) severing the bonds between the PM adaptor proteins Sla2 and Ent1 and the actin cytoskeleton and (2) hindering recruitment of Rvs167, an N-BAR–containing protein important for vesicle fission to endocytosis sites. These results underline the importance of biophysical cues in the regulation of cellular and molecular processes.

The endoribonuclease Regnase-1 suppresses inflammation through RNA degradation. Here, we show that Regnase-1 is phosphorylated and inactivated by the Act1-TBK1-IKKi axis during IL-17 stimulation. Moreover, this phosphorylation substantially contributes to the mRNA stabilization needed for amplification of TH17-cell–mediated inflammation., Regnase-1 (also known as Zc3h12a or MCPIP-1) is an endoribonuclease involved in mRNA degradation of inflammation-associated genes. Regnase-1 is inactivated in response to external stimuli through post-translational modifications including phosphorylation, yet the precise role of phosphorylation remains unknown. Here, we demonstrate that interleukin (IL)-17 induces phosphorylation of Regnase-1 in an Act1-TBK1/IKKi–dependent manner, especially in nonhematopoietic cells. Phosphorylated Regnase-1 is released from the endoplasmic reticulum (ER) into the cytosol, thereby losing its mRNA degradation function, which leads to expression of IL-17 target genes. By using CRISPR/Cas-9 technology, we generated Regnase-1 mutant mice, in which IL-17–induced Regnase-1 phosphorylation is completely blocked. Mutant mice (Regnase-1AA/AA and Regnase-1ΔCTD/ΔCTD) were resistant to the IL-17–mediated inflammation caused by T helper 17 (Th17) cells in vivo. Thus, Regnase-1 plays a critical role in the development of IL-17–mediated inflammatory diseases via the Act1-TBK1-IKKi axis, and blockade of Regnase-1 phosphorylation sites may be promising for treatment of Th17-associated diseases., Graphical Abstract